Synergistic Bacterial Compositions and Methods of Production and Use Thereof

ABSTRACT

Provided are therapeutic compositions containing Ecobiotic™ populations for prevention, treatment and reduction of symptoms associated with a dysbiosis of a mammalian subject such as a human.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/223,008, filed Dec. 17, 2018 (currently allowed), which is acontinuation of U.S. patent application Ser. No. 15/359,439, filed Nov.22, 2016 (abandoned), which is a continuation of U.S. patent applicationSer. No. 14/592,481, filed Jan. 8, 2015 (now U.S. Pat. No. 9,533,014,issued on Jan. 3, 2017), which is a continuation of U.S. patentapplication Ser. No. 14/221,190, filed Mar. 20, 2014 (now U.S. Pat. No.9,028,841, issued on May 12, 2015), which is a divisional of U.S. patentapplication Ser. No. 14/091,201, filed on Nov. 26, 2013 (now U.S. Pat.No. 8,906,668, issued on Dec. 9, 2014), which is a continuation ofInternational Application No. PCT/US2013/071758, filed on Nov. 25, 2013,which claims the benefit of U.S. Provisional Patent Application No.61/729,518, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,519, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,520, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,521, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,522, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,524, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,515, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,517, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,525, filed Nov. 23, 2012, U.S. Provisional Patent Application No.61/729,526, filed Nov. 23, 2012, and U.S. Provisional Patent ApplicationNo. 61/729,527, filed Nov. 23, 2012, all of which are incorporatedherein by reference in their entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 4268_024000K_Seqlisting_ST25.txt; Size: 3,949,067bytes; and Date of Creation: Dec. 14, 2020) filed with the applicationis herein incorporated by reference in its entirety.

INTRODUCTION

Mammals are colonized by microbes in the gastrointestinal (GI) tract, onthe skin, and in other epithelial and tissue niches such as the oralcavity, eye surface and vagina. The gastrointestinal tract harbors anabundant and diverse microbial community. It is a complex system,providing an environment or niche for a community of many differentspecies or organisms, including diverse strains of bacteria. Hundreds ofdifferent species may form a commensal community in the GI tract in ahealthy person, and this complement of organisms evolves from the timeof birth to ultimately form a functionally mature microbial populationby about 3 years of age. Interactions between microbial strains in thesepopulations and between microbes and the host, e.g. the host immunesystem, shape the community structure, with availability of andcompetition for resources affecting the distribution of microbes. Suchresources may be food, location and the availability of space to grow ora physical structure to which the microbe may attach. For example, hostdiet is involved in shaping the GI tract flora.

A healthy microbiota provides the host with multiple benefits, includingcolonization resistance to a broad spectrum of pathogens, essentialnutrient biosynthesis and absorption, and immune stimulation thatmaintains a healthy gut epithelium and an appropriately controlledsystemic immunity. In settings of ‘dysbiosis’ or disrupted symbiosis,microbiota functions can be lost or deranged, resulting in increasedsusceptibility to pathogens, altered metabolic profiles, or induction ofproinflammatory signals that can result in local or systemicinflammation or autoimmunity. Thus, the intestinal microbiota plays asignificant role in the pathogenesis of many diseases and disorders,including a variety of pathogenic infections of the gut. For instance,patients become more susceptible to pathogenic infections when thenormal intestinal microbiota has been disturbed due to use ofbroad-spectrum antibiotics. Many of these diseases and disorders arechronic conditions that significantly decrease a patient's quality oflife and can be ultimately fatal.

Fecal transplantation has been shown to be an effective treatment forpatients suffering from severe or refractory GI infections byrepopulating the gut with a diverse array of microbes that control keypathogens by creating an ecological environment inimical to theirproliferation and survival. Such approaches have demonstratedsignificant potential to decrease host susceptibility to infection.Fecal transplantation, however, is considered to be a procedure of lastresort because it has the potential to transmit infectious or allergenicagents between hosts, involves the transmission of potentially hundredsof unknown strains from donor to patient, and is difficult to perform ona mass scale. Additionally, fecal transplantation is inherentlynonstandardized and different desired and/or undesired material may betransmitted in any given donation. Fecal transplantation is not approvedby the FDA and is unlikely to gain approval since the product cannot bestandardized and characterized according to regulatory requirements foridentity, potency, purity and safety. Thus, there is a need for definedcompositions that can be used to decrease susceptibility to infectionand/or that facilitate restoration of a healthy gut microbiota.

Thus practitioners have a need for a much safer and reproducibletreatment for disorders currently treated on an experimental (non-FDAapproved) basis using fecal transplantation. In order to prepare atherapeutic with commercial potential, we have designed bacterialcompositions of isolated bacterial strains with a plurality ofbeneficial properties based on our understanding of those bacterialstrains and our analysis of the properties that would enhance theutility and commercialization of a bacterial composition.

Therefore, in response to the need for durable, efficient, and effectivecompositions and methods for treatment of GI diseases, in particularserious pathogenic infections, by way of restoring or enhancingmicrobiota functions, we address these and other shortcomings of theprior art by providing compositions and methods for treating patients.

SUMMARY

In one aspect, provided are compositions comprising an effective amountof a bacterial composition comprising at least a first type of isolatedbacterium capable of forming a spore and a second type of isolatedbacterium capable of forming a spore, wherein the first type and thesecond type are not identical, and wherein at least one of the firsttype and the second type are capable of decreasing and/or inhibiting thegrowth and/or colonization of at least one type of pathogenic bacteria.In an embodiment, the bacterial composition comprises at least about 3,4, 5, 6, 7, 8, 9, or 10 types of isolated bacteria. In an embodiment,the bacterial composition comprises at least about 3, 4, 5, 6, 7, 8, 9,or 10 types of isolated bacteria and at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 of the isolated bacteria are capable of forming spores. In anembodiment, the bacterial composition comprises at least about 5 typesof isolated bacteria and at least 2 of the isolated bacteria are capableof forming spores. In an embodiment, the bacterial compositioncomprises: i) at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or moretypes of isolated bacteria capable of forming spores, ii) at least about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more types of isolated bacteria notknown to be capable of forming spores, or iii) any combination of i) andii). In an embodiment, the first type and the second type are present inthe composition in approximately equal concentrations. In an embodiment,the first type and the second type are present in the composition in notsubstantially equal concentrations. In an embodiment, the first type ispresent in the composition in at least about 150% the concentration ofthe second type, or wherein the second type is present in thecomposition in at least about 150% the concentration of the first type.In an embodiment, the composition consists essentially of: i) betweentwo and about twenty types of isolated bacteria, wherein at least twotypes of isolated bacteria are independently capable of spore formation;ii) between two and about twenty types of isolated bacteria, wherein atleast two types of isolated bacteria not known to be capable of sporeformation, or iii) any combination of i) and ii). In an embodiment, thefirst type of isolated bacterium and the second type of isolatedbacterium are selected from Table 1. In an embodiment, the first type ofisolated bacterium and the second type of isolated bacterium comprise anoperational taxonomic unit (OTU) distinction. In an embodiment, the OTUdistinction comprises 16S rRNA sequence similarity below about 95%identity. In an embodiment, the first type of isolated bacterium and thesecond type of isolated bacterium independently comprise bacteria thatcomprise 16S rRNA sequence at least 95% identical to 16S rRNA sequencepresent in a bacterium selected from SEQ ID NOs.: 1-1,864. In anembodiment, a combination of the first type and the second type are: i)cytotoxic, ii) cytostatic, iii) capable of decreasing the growth of thepathogenic bacterium, iv) capable of inhibiting the growth of thepathogenic bacterium, v) capable of decreasing the colonization of thepathogenic bacterium, vi) capable of inhibiting the colonization of thepathogenic bacterium, or vii) any combination of i)-vi). In anembodiment, the combination is capable of inhibiting proliferation ofthe pathogenic bacteria present at a concentration at least equal to theconcentration of the combination of the first type and the second type.In an embodiment, the combination is capable of inhibiting proliferationof the pathogenic bacterial present at a concentration at least abouttwice the concentration of the combination of the first type and thesecond type. In an embodiment, the combination is capable of inhibitingproliferation of the pathogenic bacterial present at a concentration atleast about ten times the concentration of the combination of the firsttype and the second type. In an embodiment, the pathogenic bacterium isselected from the group consisting of Yersinia, Vibrio, Treponema,Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia,Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria,Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter,Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus,multi-drug resistant bacteria, Carbapenem-resistent Enterobacteriaceae(CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), andvancomycin-resistant Enterococci (VRE). In an embodiment, the first typeand the second type synergistically interact. In an embodiment, thefirst type and the second type synergistically interact to inhibit thepathogenic bacterium.

In another aspect, provided are compositions comprising an effectiveamount of a bacterial composition comprising at least a first type ofisolated bacterium and a second type of isolated bacterium, wherein onlyone of the first type and the second type are capable of forming aspore, and wherein at least one of the first type and the second typeare capable of decreasing the growth and/or colonization of at least onetype of pathogenic bacteria.

In another aspect, provided are compositions comprising an effectiveamount of a bacterial composition comprising at least a first type ofisolated bacterium and a second type of isolated bacterium, wherein thefirst type and the second type are not spores or known to be capable offorming a spore, and wherein at least one of the first type and thesecond type are capable of decreasing the growth and/or colonization ofat least one type of pathogenic bacteria.

In an embodiment, at least one of the first type and the second type arecapable of reducing the growth rate of at least one type of pathogenicbacteria. In an embodiment, at least one of the first type and thesecond type are cytotoxic to at least one type of pathogenic bacteria.In an embodiment, at least one of the first type and the second type arecytostatic to at least one type of pathogenic bacteria. In anembodiment, the first type and the second type are selected fromTable 1. In an embodiment, the first type and the second type comprisedifferent species. In an embodiment, the first type and the second typecomprise different genera. In an embodiment, the first type and thesecond type comprise different families. In an embodiment, the firsttype and the second type comprise different orders.

In another aspect, provided are compositions comprising an effectiveamount of a bacterial composition comprising at least a first type ofisolated bacterium and a second type of isolated bacterium, wherein: i)the first type and the second type are independently capable of forminga spore; ii) only one of the first type and the second type are capableof forming a spore or iii) neither the first type nor the second typeare capable of forming a spore, wherein the first type and the secondtype are not identical, wherein the first type and the second type arecapable of functionally populating the gastrointestinal tract of a humansubject to whom the composition is administered. In an embodiment, thefunctional populating of the gastrointestinal tract comprises preventinga dysbiosis of the gastrointestinal tract. In an embodiment, thefunctional populating of the gastrointestinal tract comprises treating adysbiosis of the gastrointestinal tract. In an embodiment, thefunctional populating of the gastrointestinal tract comprises reducingthe severity of a dysbiosis of the gastrointestinal tract. In anembodiment, the functional populating of the gastrointestinal tractcomprises reducing one or more symptoms of a dysbiosis of thegastrointestinal tract. In an embodiment, the functional populating ofthe gastrointestinal tract comprises preventing growth and/orcolonization of the gastrointestinal tract by a pathogenic bacterium. Inan embodiment, the functional populating of the gastrointestinal tractcomprises reducing growth and/or colonization of the gastrointestinaltract by a pathogenic bacterium. In an embodiment, the functionalpopulating of the gastrointestinal tract comprises reducing the numberof one or more types of pathogenic bacteria in the gastrointestinaltract. In an embodiment, the functional populating of thegastrointestinal tract comprises increasing the number of one or morenon-pathogenic bacteria in the gastrointestinal tract. In an embodiment,the bacterial composition comprises 0, 1, 2, 3 or greater than 3 typesof isolated bacteria capable of forming spores. In an embodiment, thebacterial composition comprises at least about 5 types of isolatedbacteria capable of forming spores. In an embodiment, the bacterialcomposition comprises at least about 7 types of isolated bacteriacapable of forming spores. In an embodiment, the first type and thesecond type are present in the composition in not substantially equalconcentrations. In an embodiment, the first type and the second type arepresent in the composition in approximately equal concentrations. In anembodiment, the first type is present in the composition in at leastabout 150% the concentration of the second type. In an embodiment, thesecond type is present in the composition in at least about 150% theconcentration of the first type. In an embodiment, the compositionconsists essentially of between two and about ten types of isolatedbacteria, wherein at least one type of isolated bacteria areindependently capable of spore formation. In an embodiment, the firsttype of isolated bacterium and the second type of isolated bacterium areselected from Table 1. In an embodiment, the first type of isolatedbacterium and the second type of isolated bacterium comprise anoperational taxonomic unit (OTU) distinction. In an embodiment, the OTUdistinction comprises 16S rRNA sequence similarity below about 95%identity. In an embodiment, the first type of isolated bacterium and thesecond type of isolated bacterium independently comprise bacteria thatcomprise 16S rRNA sequence at least 95% identical to 16S rRNA sequencepresent in a bacterium selected from Table 3. In an embodiment, acombination of the first type and the second type are cytotoxic orcytostatic to the pathogenic bacterium. In an embodiment, thecombination is capable of inhibiting proliferation of the pathogenicbacteria present at a concentration at least equal to the concentrationof the combination of the first type and the second type. In anembodiment, the combination is capable of inhibiting proliferation ofthe pathogenic bacterial present at a concentration at least about twicethe concentration of the combination of the first type and the secondtype. In an embodiment, the combination is capable of inhibitingproliferation of the pathogenic bacteria present at a concentration atleast about ten times the concentration of the combination of the firsttype and the second type. In an embodiment, the pathogenic bacterium isselected from the group consisting of Yersinia, Vibrio, Treponema,Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia,Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria,Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter,Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus,multi-drug resistant bacteria, Carbapenem-resistent Enterobacteriaceae(CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), andvancomycin-resistant Enterococci (VRE). In an embodiment, the first typeand the second type synergistically interact to be cytotoxic to thepathogenic bacterium. In an embodiment, wherein the first type and thesecond type synergistically interact to be cytostatic to the pathogenicbacterium.

In another aspect, provided are single dose units comprising thecompositions of the present invention. In an embodiment, the dose unitcomprises at least 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹ or greater than 1×10¹¹ colony forming units (CFUs) of eitherspores or vegetative bacterial cells. In an embodiment, the dose unitcomprises a pharmaceutically acceptable excipient, an enteric coating ora combination thereof. In an embodiment, the dose unit further comprisesa drug selected from corticosteroids, mesalazine, mesalamine,sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs,cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate,antihistamines, glucocorticoids, epinephrine, theophylline, cromolynsodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis,anti-cholinergic decongestants, mast-cell stabilizers, monoclonalanti-IgE antibodies, vaccines, and combinations thereof. In anembodiment, the dose unit is formulated for oral administration, rectaladministration, or the combination of oral and rectal administration, oris formulated for topical, nasal or inhalation administration.

In another aspect, provided are kits comprising in one or morecontainers: a first purified population of a first type of bacterialspores substantially free of viable vegetal bacterial cells; and asecond purified population of a second type of bacterial sporessubstantially free of viable vegetal bacterial cells, wherein the firsttype and the second type of bacterial spores are not identical, andwherein the first type and the second type of bacterial spores, whenco-localized in a target region of a gastrointestinal tract of a humansubject in need thereof, are capable of functionally populating thegastrointestinal tract. In an embodiment, the first purified populationand the second purified population are present in a single container. Inan embodiment, the first purified population and the second purifiedpopulation are present in two containers. In an embodiment, the firstpurified population and the second purified population are lyophilizedor substantially dehydrated. In an embodiment, the kit further comprisesin one or more containers an effective amount of an anti-bacterialagent, an effective amount of an anti-viral agent, an effective amountof an anti-fungal agent, an effective amount of an anti-parasitic agent,or a combination thereof in one or more containers. In an embodiment,the kit further comprises a pharmaceutically acceptable excipient ordiluent.

Also provided are pharmaceutical formulations comprising an effectiveamount of the compositions of the invention, and further comprising aneffective amount of an anti-bacterial agent, an effective amount of ananti-fungal agent, an effective amount of an anti-viral agent, aneffective amount of an anti-parasitic agent.

Also provided are comestible products comprising a first purifiedpopulation of a first type of bacterial spores and a second purifiedpopulation of a second type of bacterial spores, wherein the first typeand the second type of bacterial spores are not identical, wherein thecomestible product is substantially free of viable vegetal bacterialcells, and wherein the first type and the second type of bacterialspores, when administered to a human subject in need thereof, arecapable of functionally populating the gastrointestinal tract of thehuman subject. In an embodiment, the comestible product comprises a foodor food additive, a beverage or beverage additive, or a medical food. Inan embodiment, the comestible product comprises at least 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹ or greater than 1×10¹¹ colonyforming units (CFUs) of viable spores. In an embodiment, the comestibleproduct comprises a first type of bacterial spores and a second type ofbacterial spores selected from Table 1, or where the first type ofbacterial spores and the second type of bacterial spores independentlycomprise bacterial spores that comprise 16S rRNA sequence at least 95%identical to 16S rRNA sequence present in a bacterium selected from SEQID NOs.: 1-1,864.

Also provided are methods comprising administering to a human subject inneed thereof an effective amount of a bacterial composition comprisingat least a first type of isolated bacterium and a second type ofisolated bacterium, wherein: the first type and the second type areindependently capable of forming a spore; only one of the first type andthe second type are capable of forming a spore or neither the first typenor the second type are capable of forming a spore, wherein the firsttype and the second type are not identical, and wherein at least one ofthe first type and the second type exert an inhibitory-effect on apathogenic bacterium present in the gastrointestinal tract of the humansubject, such that the number of pathogenic bacteria present in thegastrointestinal tract is not detectably increased or is detectablydecreased over a period of time. In an embodiment, the human subject isdiagnosed as having a dysbiosis of the gastrointestinal tract. In anembodiment, wherein the human subject is diagnosed as infected with apathogenic bacterium selected from the group consisting of Yersinia,Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella, Salmonella,Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium,Listeria, Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter,Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus,multi-drug resistant bacteria, Carbapenem-resistent Enterobacteriaceae(CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), andvancomycin-resistant Enterococci (VRE). In an embodiment, the bacterialcomposition is administered simultaneously with i) an antibiotic, ii) aprebiotic, or iii) a combination of i) and ii). In an embodiment, thebacterial composition is administered prior to administration of i) anantibiotic, ii) a prebiotic, or iii) a combination of i) and ii). In anembodiment, the bacterial composition is administered subsequent toadministration of i) an antibiotic, ii) a prebiotic, or iii) acombination of i) and ii). In an embodiment, the number of pathogenicbacterium present in or excreted from the gastrointestinal tract of thehuman subject is detectably reduced within one month, within two weeks,or within one week of administration of the bacterial composition. In anembodiment, the number of pathogenic bacterium present in or excretedfrom the gastrointestinal tract of the human subject is detectablyreduced within three days, two days or one day of administration of thebacterial composition. In an embodiment, the human subject is detectablyfree of the pathogenic bacterium within one month, two weeks, one week,three days or one day of administration of the bacterial composition. Inan embodiment, the bacterial composition comprises at least about 3, 4,5, 6, 7, 8, 9, or 10 types of isolated bacteria. In an embodiment, thebacterial composition comprises at least about 3, 4, 5, 6, 7, 8, 9, or10 types of isolated bacteria and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 of the isolated bacteria are capable of forming spores. In anembodiment, the bacterial composition comprises at least about 5 typesof isolated bacteria and at least 2 of the isolated bacteria are capableof forming spores. In an embodiment, the bacterial compositioncomprises: i) at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or moretypes of isolated bacteria capable of forming spores, ii) at least about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more types of isolated bacteria notknown to be capable of forming spores, or iii) any combination of i) andii). In an embodiment, the bacterial composition comprises at leastabout 5 types of isolated bacteria and at least 1 of the isolatedbacteria are capable of forming spores. In an embodiment, the bacterialcomposition comprises at least about 5 types of isolated bacteria and atleast 1 of the isolated bacteria is not capable of forming spores. In anembodiment, the bacterial composition comprises at least about 3, 4, 5,6, 7, 8, 9 or 10 types of isolated bacteria, wherein i) at least 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 types of isolated bacteria are capable offorming spores, ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types ofisolated bacteria are not capable of forming spores, or iii) anycombination of i) and ii). In an embodiment, the first type and thesecond type are present in the composition in approximately equalconcentrations. In an embodiment, the first type and the second type arepresent in the composition in not substantially equal concentrations. Inan embodiment, the first type is present in the composition in at leastabout 150% the concentration of the second type, or wherein the secondtype is present in the composition in at least about 150% theconcentration of the first type. In an embodiment, the compositionconsists essentially of between two and about ten types of isolatedbacteria, wherein at least two types of isolated bacteria areindependently capable of spore formation. In an embodiment, thecomposition consists essentially of between two and about ten types ofisolated bacteria, wherein at least two types of isolated bacteria arenot capable of spore formation. In an embodiment, the first type ofisolated bacterium and the second type of isolated bacterium areselected from Table 1. In an embodiment, the first type of isolatedbacterium and the second type of isolated bacterium comprise anoperational taxonomic unit (OTU) distinction. In an embodiment, the OTUdistinction comprises 16S rRNA sequence similarity below about 95%identity. In an embodiment, the first type of isolated bacterium and thesecond type of isolated bacterium independently comprise bacteria thatcomprise 16S rRNA sequence at least 95% identical to 16S rRNA sequencepresent in a bacterium selected from SEQ ID NOs: 1-1,864. In anembodiment, a combination of the first type and the second type arecytotoxic or cytostatic to the pathogenic bacterium. In an embodiment,the combination is capable of inhibiting proliferation of the pathogenicbacterial present at a concentration at least equal to the concentrationof the combination of the first type and the second type. In anembodiment, the combination is capable of inhibiting proliferation ofthe pathogenic bacterial present at a concentration at least about twicethe concentration of the combination of the first type and the secondtype. In an embodiment, the combination is capable of inhibitingproliferation of the pathogenic bacterial present at a concentration atleast about ten times the concentration of the combination of the firsttype and the second type. In an embodiment, the pathogenic bacterium isselected from the group consisting of Yersinia, Vibrio, Treponema,Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia,Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria,Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter,Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus,multi-drug resistant bacteria, Carbapenem-resistent Enterobacteriaceae(CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), andvancomycin-resistant Enterococci (VRE). In an embodiment, the first typeand the second type synergistically interact to be cytotoxic to thepathogenic bacterium. In an embodiment, the first type and the secondtype synergistically interact to be cytostatic to the pathogenicbacterium.

Also provided are methods of functionally populating thegastrointestinal tract of a human subject, comprising administering tothe subject an effective amount of a bacterial composition comprising atleast a first type of isolated bacterium and a second type of isolatedbacterium, wherein i) the first type and the second type areindependently capable of forming a spore; ii) only one of the first typeand the second type are capable of forming a spore or iii) neither thefirst type nor the second type are capable of forming a spore, whereinthe first type and the second type are not identical, under conditionssuch that the first type and the second type functionally populate thegastrointestinal tract of the human subject. In an embodiment, thebacterial composition is orally administered, rectally administered, orthe combination of orally and rectally administered. In an embodiment,the bacterial composition is topically or nasally administered orinhaled. In an embodiment, the first type of isolated bacteria and thesecond type of isolated bacteria are selected from Table 1. In anembodiment, the bacterial composition consists essentially of spores,wherein the spores comprise spores of the first type of isolatedbacteria and spores of the second type of isolated bacteria. In anembodiment, the first type of isolated bacteria and the second type ofisolated bacteria independently comprise bacterial spores that comprise16S rRNA sequence at least 95% identical to 16S rRNA sequence present ina bacterium selected from SEQ ID NOs. 1-1,864. In an embodiment, thefunctional populating of the gastrointestinal tract comprises preventinga dysbiosis of the gastrointestinal tract. In an embodiment, thefunctional populating of the gastrointestinal tract comprises treating adysbiosis of the gastrointestinal tract. In an embodiment, thefunctional populating of the gastrointestinal tract comprises reducingthe severity of a dysbiosis of the gastrointestinal tract. In anembodiment, the functional populating of the gastrointestinal tractcomprises reducing one or more symptoms of a dysbiosis of thegastrointestinal tract. In an embodiment, the functional populating ofthe gastrointestinal tract comprises preventing colonization of thegastrointestinal tract by a pathogenic bacterium. In an embodiment, thefunctional populating of the gastrointestinal tract comprises reducingcolonization of the gastrointestinal tract and/or growth by a pathogenicbacterium. In an embodiment, wherein the functional populating of thegastrointestinal tract comprises reducing the number of one or moretypes of pathogenic bacteria in the gastrointestinal tract. In anembodiment, the functional populating of the gastrointestinal tractcomprises increasing the number of one or more non-pathogenic bacteriain the gastrointestinal tract. In an embodiment, the bacterialcomposition comprises at least about 3, 5, 7 or 9 types of isolatedbacteria capable of forming spores. In an embodiment, the bacterialcomposition comprises at least about 5 types of isolated bacteria and atleast 20% of the isolated bacteria are capable of forming spores. In anembodiment, the bacterial composition comprises at least about 5 typesof isolated bacteria and at least 2 of the isolated bacteria are capableof forming spores. In an embodiment, the bacterial composition comprisesat least about 3, 4, 5, 6, 7, 8, 9 or 10 types of isolated bacteria,wherein i) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types of isolatedbacteria are capable of forming spores, ii) at least 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 types of isolated bacteria are not capable of formingspores, or iii) any combination of i) and ii). In an embodiment, thefirst type and the second type are present in the composition inapproximately equal concentrations. In an embodiment, the first type andthe second type are present in the composition in not substantiallyequal concentrations. In an embodiment, the first type is present in thecomposition in at least about 150% the concentration of the second type,or wherein the second type is present in the composition in at leastabout 150% the concentration of the first type. In an embodiment, thecomposition consists essentially of between two and about ten types ofisolated bacteria, wherein i) at least one type of isolated bacteria iscapable of spore formation, ii) at least one type of isolated bacteriais not capable of spore formation, or iii) a combination of i) and ii).In an embodiment, a combination of the first type and the second typeare inhibitory to the pathogenic bacterium. In an embodiment, thecombination reduces the growth rate of the pathogenic bacterium. In anembodiment, the combination is cytostatic or cytotoxic to the pathogenicbacterium. In an embodiment, the combination is capable of inhibitinggrowth of the pathogenic bacterial present at a concentration at leastequal to the concentration of the combination of the first type and thesecond type. In an embodiment, the combination is capable of inhibitinggrowth of the pathogenic bacterial present at a concentration at leastabout twice the concentration of the combination of the first type andthe second type. In an embodiment, the combination is capable ofinhibiting proliferation of the pathogenic bacterial present at aconcentration at least about ten times the concentration of thecombination of the first type and the second type. In an embodiment, thepathogenic bacterium is selected from the group consisting of Yersinia,Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella, Salmonella,Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium,Listeria, Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter,Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus,multi-drug resistant bacteria, Carbapenem-resistent Enterobacteriaceae(CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), andvancomycin-resistant Enterococci (VRE). In an embodiment, the first typeand the second type synergistically interact to reduce or inhibit thegrowth of the pathogenic bacterium. In an embodiment, the first type andthe second type synergistically interact to reduce or inhibit thecolonization of the pathogenic bacterium. In an embodiment, the methodcomprises administering to the human subject a single dose unitcomprising at least 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹ or greater than 1×10¹¹ colony forming units (CFUs) of viablebacteria. In an embodiment, the dose unit comprises a bacterialpopulation substantially in the form of spores. In an embodiment, thedose unit comprises a pharmaceutically acceptable excipient and/or anenteric coating. In an embodiment, the unit dose is formulated for oraladministration, rectal administration, or the combination of oral andrectal administration. In an embodiment, the unit dose is formulated fortopical or nasal administration or for inhalation.

In another aspect, provided are methods of reducing the number ofpathogenic bacteria present in the gastrointestinal tract of a humansubject, comprising administering to the subject an effective amount ofa pharmaceutical formulation comprising an effective amount of thecomposition of claim 1 and further comprising an effective amount of ananti-microbial agent, under conditions such that the number ofpathogenic bacteria present in the gastrointestinal tract of the humansubject is reduced within about one month of administration of thepharmaceutical formulation. In an embodiment, the number of pathogenicbacteria present in the gastrointestinal tract of the human subject isreduced within about two weeks of administration of the pharmaceuticalformulation. In an embodiment, the number of pathogenic bacteria presentin the gastrointestinal tract of the human subject is reduced withinabout one week of administration of the pharmaceutical formulation. Inan embodiment, the number of pathogenic bacteria present in thegastrointestinal tract of the human subject is reduced within aboutthree days of administration of the pharmaceutical formulation. In anembodiment, the number of pathogenic bacteria present in thegastrointestinal tract of the human subject is reduced within about oneday of administration of the pharmaceutical formulation. In anembodiment, the anti-microbial agent comprises anti-bacterial agent. Inan embodiment, the anti-microbial agent comprises anti-fungal agent. Inan embodiment, the anti-microbial agent comprises anti-viral agent. Inan embodiment, the anti-microbial agent comprises anti-parasitic agent.

In another aspect, provided are methods of preparing a comestibleproduct, comprising combining with a comestible carrier a first purifiedpopulation comprising at least a first type of isolated bacterium and asecond purified population comprising at least a second type of isolatedbacterium, wherein: i) the first type and the second type areindependently capable of forming a spore; ii) only one of the first typeand the second type are capable of forming a spore or iii) neither thefirst type nor the second type are capable of forming a spore, whereinthe first type and the second type of bacteria are not identical,wherein the comestible product is substantially free of non-comestiblematerials. In an embodiment, at least one of the first purifiedpopulation and the second purified population consist essentially ofviable spores. In an embodiment, the first purified population and thesecond purified population consist essentially of viable spores. In anembodiment, the comestible product is substantially free of viablevegetal bacterial cells. In an embodiment, the viable spores, when thecomestible product is consumed by a human subject in need thereof, arecapable of functionally populating the gastrointestinal tract of thehuman subject. In an embodiment, the comestible product comprises a foodor food additive. In an embodiment, the comestible product comprises abeverage or beverage additive. In an embodiment, the comestible productcomprises a medical food. In an embodiment, the comestible productcomprises at least 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹ or greater than 1×10¹¹ colony forming units (CFUs) of viablespores. In an embodiment, spores are of a bacterium selected fromTable 1. In an embodiment, the first purified population and the secondpurified population independently comprise bacterial spores thatcomprise 16S rRNA sequence at least 95% identical to 16S rRNA sequencepresent in a bacterium selected from SEQ ID NOs: 1-1,864.

Also provided are methods of reducing the abundance of a pathogen in thegastrointestinal tract of a patient comprising administering thecomposition of the invention in a therapeutically effective amount andallowing the bacterial composition to compete with the pathogen in thegastrointestinal tract of a patient.

Further provided are methods of treating diarrhea comprisingadministering the composition of the invention in a therapeuticallyeffective amount and allowing the bacterial composition to reduce thediarrheal effect of a pathogen in the gastrointestinal tract of apatient. In an embodiment, the pathogen is Aeromonas hydrophila,Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus,Campylobacter jejuni, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, enteroaggregative Escherichia coli,enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli,enterotoxigenic Escherichia coli (LT or ST), Escherichia coli 0157:H7,Helicobacter pylori, Lysteria monocytogenes, Plesiomonas shigelloides,Salmonella spp., Salmonella typhi, Shigella spp., Staphylococcus spp.,Staphylococcus aureus, Vibrio spp., Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, multi-drugresistant bacteria, Carbapenem-resistent Enterobacteriaceae (CRE), andvancomycin-resistant Enterococci (VRE). In an embodiment, the pathogenis Clostridium difficile, Salmonella spp., pathogenic Escherichia coli,or vancomycin-resistant Enterococcus spp. In an embodiment, the pathogenis Clostridium difficile. In an embodiment, the composition isadministered orally.

Also provided are therapeutic compositions comprising a first purifiedbacterial population capable forming spores consisting of Collinsellaaerofaciens and a second purified bacterial population consisting of aspecies selected from Table 1 or SEQ ID NOs: 1,865-1,915, wherein atleast one of the first type and the second type are cytotoxic orcytostatic to a pathogenic bacterium. In an embodiment, a synergisticcombination of the first bacterial spore population and the secondbacterial spore population are cytotoxic or cytostatic to the pathogenicbacterium. In an embodiment, the combination is capable of inhibitingproliferation of the pathogenic bacterial present at a concentration atleast equal to the concentration of the combination of the first typeand the second type. In an embodiment, the combination is capable ofinhibiting proliferation of the pathogenic bacterial present at aconcentration at least about twice the concentration of the combinationof the first type and the second type. In an embodiment, the combinationis capable of inhibiting proliferation of the pathogenic bacterialpresent at a concentration at least about ten times the concentration ofthe combination of the first type and the second type. In an embodiment,the pathogenic bacterium is selected from the group consisting ofYersinia, Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella,Salmonella, Rickettsia, Pseudomonas, Neisseria, Mycoplasma,Mycobacterium, Listeria, Leptospira, Legionella, Helicobacter,Haemophilus, Francisella, Escherichia, Enterococcus, Corynebacterium,Clostridium, Chlamydia, Chlamydophila, Campylobacter, Brucella,Borrelia, and Bordetella. In an embodiment, the pathogenic bacterium isClostridium dificile. In an embodiment, the first bacterial sporepopulation and the second bacterial spore population synergisticallyinteract to be cytotoxic to the pathogenic bacterium. In an embodiment,the first bacterial spore population and the second bacterial sporepopulation synergistically interact to be cytostatic to the pathogenicbacterium. In an embodiment, the bacterial composition comprises atleast about 3 types of isolated bacteria capable of forming spores. Inan embodiment, the bacterial composition comprises at least about 5types of isolated bacteria capable of forming spores. In an embodiment,the first bacterial spore population and the second bacterial sporepopulation are present in the composition in approximately equalconcentrations. In an embodiment, the composition consists essentiallyof between two and about ten bacterial spore populations of isolatedbacteria.

In another aspect, provided are therapeutic compositions comprising afirst purified bacterial spore population consisting of bacteriacomprising 16S rRNA sequence at least about 97% identical to a 16S rRNAsequence present in a reference Collinsella aerofaciens OTU, and asecond purified bacterial spore population consisting of bacteriacomprising 16S rRNA sequence at least about 97% identical to a 16S rRNAsequence present in a reference bacterium listed in Table 1 or SEQ IDNOs: 1,865-1,915, wherein at least one of the first type and the secondtype are cytotoxic or cytostatic to a pathogenic bacterium. In anembodiment, a synergistic combination of the first type and the secondtype are cytotoxic or cytostatic to the pathogenic bacterium. In anembodiment, wherein the combination is capable of inhibitingproliferation of the pathogenic bacterial present at a concentration atleast equal to the concentration of the combination of the first typeand the second type. In an embodiment, the first type and the secondtype synergistically interact to be cytotoxic or cytostatic to thepathogenic bacterium. In an embodiment, the first purified bacterialspore population and the second purified bacterial spore population arecapable of functionally populating the gastrointestinal tract of a humansubject to whom the composition is administered. In an embodiment, thefunctional populating of the gastrointestinal tract comprisespreventing, treating, reducing the severity of or reducing a symptom ofa dysbiosis of the gastrointestinal tract. In an embodiment, thefunctional populating of the gastrointestinal tract comprises i)reducing the number of one or more types of pathogenic bacteria in thegastrointestinal tract; or ii) increasing the number of one or morenon-pathogenic bacteria in the gastrointestinal tract. In an embodiment,the composition further comprises an effective amount of ananti-bacterial agent, an anti-fungal agent, an anti-viral agent or ananti-parasitic agent.

Also provided are methods of treating or preventing a recurrence of aClostridium difficile infection, comprising administering to a humansubject in need thereof an effective amount of the therapeuticcomposition of the invention under conditions such that the firstpurified bacterial spore population and the second purified bacterialspore population exert a cytotoxic or cytostatic effect on a pathogenicbacterium present in the gastrointestinal tract of the human subject,such that the number of Clostridium difficile bacteria present in thegastrointestinal tract is not detectably increased or is detectablydecreased over a period of time. In an embodiment, the number ofClostridium difficile bacteria present in or excreted from thegastrointestinal tract of the human subject is detectably reduced withinone month of administration of the bacterial composition. In anembodiment, the first purified bacterial spore population and the secondpurified bacterial spore population synergistically interact to becytotoxic and/or cytostatic to the Clostridium difficile bacteria. In anembodiment, the therapeutic composition is orally administered. In anembodiment, the therapeutic composition comprises a medical food.

In another aspect, provided are kits comprising in one or morecontainers: a first purified population of a first type of bacteriacapable of forming spores; and a second purified population of a secondtype of bacteria capable of forming spores, wherein the first type andthe second type are not identical, and wherein the first type and thesecond type, when co-localized in a target region of a gastrointestinaltract of a human subject in need thereof, are capable of functionallypopulating the gastrointestinal tract. In an embodiment, the firstpurified population and the second purified population are present in asingle container. In an embodiment, the kit is formulated for use as anutritional supplement and optionally comprising a prebiotic material.

Additional objects and advantages will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the embodiments. Theobjects and advantages will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description, serve to further explain the embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic of 16S rRNA gene and denotes thecoordinates of hypervariable regions 1-9 (V1-V9). Coordinates of V1-V9are 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173,1243-1294, and 1435-1465 respectively, based on numbering using E. colisystem of nomenclature defined by Brosius et al., Complete nucleotidesequence of a 16S ribosomal RNA gene (16S rRNA) from Escherichia coli,PNAS 75(10):4801-4805 (1978). FIG. 1B highlights in bold the nucleotidesequences for each hypervariable region in the exemplary reference E.coli 16S sequence described by Brosius et al. FIG. 1B discloses SEQ IDNO: 1926.

BRIEF DESCRIPTION OF TABLES

Table 1 provides bacterial species and Operational Taxonomic Units(OTUs) of the bacterial compositions of the present invention, includingtaxonometric status and the ability of the OTU to form a viable spore asprovided herein.

Table 2 provides representative combinations of the bacterialcompositions of the present invention.

16S rRNA sequences of the bacterial species and Operational TaxonomicUnits (OTUs) of the bacterial compositions of the present invention areprovided in the CRF version of the Sequence Listing as SEQ ID NOS1-1,864.

The taxonometric status, exemplary phylogenetic surrogacy and 16S rRNAsequences of exemplary bacterial compositions of the present inventionare provided in the CRF version of the Sequence Listing as SEQ ID NOS1,865-1,915.

Table 3 demonstrates the efficacy of exemplary bacterial compositions ofthe present invention in inhibiting a pathogenic bacterium.

Table 4 demonstrates the efficacy of exemplary bacterial compositions ofthe present invention in inhibiting a pathogenic bacterium.

Table 5 provides representative bacterial pathogens.

Table 6 provides representative human diseases, disorders and conditionsfor which the provided bacterial compositions are useful.

Table 7 provides representative human diseases, disorders and conditionsfor which the provided bacterial compositions are useful.

Definitions

“Microbiota” refers to the communities of microbes that live in or onthe patient's body, both sustainably and transiently, includingeukaryotes, archaea, bacteria, and viruses (including bacterial viruses(i.e., phage)).

“Dysbiosis” refers to a state of the microbiota or microbiome of the gutor other body area, including mucosal or skin surfaces in which thenormal diversity and/or function of the ecological network is disrupted.Any disruption from the preferred (e.g., ideal) state of the microbiotacan be considered a dysbiosis, even if such dysbiosis does not result ina detectable decrease in health. This state of dysbiosis may beunhealthy, it may be unhealthy under only certain conditions, or it mayprevent a subject from becoming healthier. Dysbiosis may be due to adecrease in diversity, the overgrowth of one or more pathogens orpathobionts, symbiotic organisms able to cause disease only when certaingenetic and/or environmental conditions are present in a patient, or theshift to an ecological network that no longer provides a beneficialfunction to the host and therefore no longer promotes health.

A “spore” or a population of “spores” includes bacteria (or othersingle-celled organisms) that are generally viable, more resistant toenvironmental influences such as heat and bacteriocidal agents thanvegetative forms of the same bacteria, and typically capable ofgermination and out-growth. “Spore-formers” or bacteria “capable offorming spores” are those bacteria containing the genes and othernecessary abilities to produce spores under suitable environmentalconditions.

The terms “pathogen”, “pathobiont” and “pathogenic” in reference to abacterium or any other organism or entity includes any such organism orentity that is capable of causing or affecting a disease, disorder orcondition of a host organism containing the organism or entity.

The term “isolated” encompasses a bacterium or other entity or substancethat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in nature or inan experimental setting), and/or (2) produced, prepared, purified,and/or manufactured by the hand of man. Isolated bacteria may beseparated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated bacteria are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. As usedherein, a substance is “pure” if it is substantially free of othercomponents. The terms “purify,” “purifying” and “purified” refer to abacterium or other material that has been separated from at least someof the components with which it was associated either when initiallyproduced or generated (e.g., whether in nature or in an experimentalsetting), or during any time after its initial production. A bacteriumor a bacterial population may be considered purified if it is isolatedat or after production, such as from a material or environmentcontaining the bacterium or bacterial population, and a purifiedbacterium or bacterial population may contain other materials up toabout 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, or above about 90% and still be considered“isolated.” In some embodiments, purified bacteria and bacterialpopulations are more than about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or more than about 99% pure. In the instance ofbacterial compositions provided herein, the one or more bacterial typespresent in the composition can be independently purified from one ormore other bacteria produced and/or present in the material orenvironment containing the bacterial type. Bacterial compositions andthe bacterial components thereof are generally purified from residualhabitat products.

“Inhibition” of a pathogen encompasses the inhibition of any desiredfunction or activity of the bacterial compositions of the presentinvention. Demonstrations of pathogen inhibition, such as decrease inthe growth of a pathogenic bacterium or reduction in the level ofcolonization of a pathogenic bacterium are provided herein and otherwiserecognized by one of ordinary skill in the art. Inhibition of apathogenic bacterium's “growth” may include inhibiting the increase insize of the pathogenic bacterium and/or inhibiting the proliferation (ormultiplication) of the pathogenic bacterium. Inhibition of colonizationof a pathogenic bacterium may be demonstrated by measuring the amount orburden of a pathogen before and after a treatment. An “inhibition” orthe act of “inhibiting” includes the total cessation and partialreduction of one or more activities of a pathogen, such as growth,proliferation, colonization, and function.

The “colonization” of a host organism includes the non-transitoryresidence of a bacterium or other microscopic organism. As used herein,“reducing colonization” of a host subject's gastrointestinal tract (orany other microbiotal niche) by a pathogenic bacterium includes areduction in the residence time of the pathogen in the gastrointestinaltract as well as a reduction in the number (or concentration) of thepathogen in the gastrointestinal tract or adhered to the luminal surfaceof the gastrointestinal tract. Measuring reductions of adherentpathogens may be demonstrated, e.g., by a biopsy sample, or reductionsmay be measured indirectly, e.g., by measuring the pathogenic burden inthe stool of a mammalian host.

A “combination” of two or more bacteria includes the physicalco-existence of the two bacteria, either in the same material or productor in physically connected products, as well as the temporalco-administration or co-localization of the two bacteria.

A “cytotoxic” activity or bacterium includes the ability to kill abacterial cell, such as a pathogenic bacterial cell. A “cytostatic”activity or bacterium includes the ability to inhibit, partially orfully, growth, metabolism, and/or proliferation of a bacterial cell,such as a pathogenic bacterial cell.

To be free of “non-comestible products” means that a bacterialcomposition or other material provided herein does not have asubstantial amount of a non-comestible product, e.g., a product ormaterial that is inedible, harmful or otherwise undesired in a productsuitable for administration, e.g., oral administration, to a humansubject. Non-comestible products are often found in preparations ofbacteria from the prior art.

“Microbiome” refers to the genetic content of the communities ofmicrobes that live in and on the human body, both sustainably andtransiently, including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses (i.e., phage)), wherein “genetic content”includes genomic DNA, RNA such as micro RNA and ribosomal RNA, theepigenome, plasmids, and all other types of genetic information.

“Residual habitat products” refers to material derived from the habitatfor microbiota within or on a human or animal. For example, microbiotalive in feces in the gastrointestinal tract, on the skin itself, insaliva, mucus of the respiratory tract, or secretions of thegenitourinary tract (i.e., biological matter associated with themicrobial community). Substantially free of residual habitat productsmeans that the bacterial composition no longer contains the biologicalmatter associated with the microbial environment on or in the human oranimal subject and is 100% free, 99% free, 98% free, 97% free, 96% free,or 95% free of any contaminating biological matter associated with themicrobial community. Residual habitat products can include abioticmaterials (including undigested food) or it can include unwantedmicroorganisms. Substantially free of residual habitat products may alsomean that the bacterial composition contains no detectable cells from ahuman or animal and that only microbial cells are detectable. In oneembodiment, substantially free of residual habitat products may alsomean that the bacterial composition contains no detectable viral(including bacterial viruses (i.e., phage)), fungal, mycoplasmalcontaminants. In another embodiment, it means that fewer than 1×10⁻²%,1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸ of the viable cellsin the bacterial composition are human or animal, as compared tomicrobial cells. There are multiple ways to accomplish this degree ofpurity, none of which are limiting. Thus, contamination may be reducedby isolating desired constituents through multiple steps of streaking tosingle colonies on solid media until replicate (such as, but not limitedto, two) streaks from serial single colonies have shown only a singlecolony morphology. Alternatively, reduction of contamination can beaccomplished by multiple rounds of serial dilutions to single desiredcells (e.g., a dilution of 10⁻⁸ or 10⁻⁹), such as through multiple10-fold serial dilutions. This can further be confirmed by showing thatmultiple isolated colonies have similar cell shapes and Gram stainingbehavior. Other methods for confirming adequate purity include geneticanalysis (e.g. PCR, DNA sequencing), serology and antigen analysis,enzymatic and metabolic analysis, and methods using instrumentation suchas flow cytometry with reagents that distinguish desired constituentsfrom contaminants.

“Phylogenetic tree” refers to a graphical representation of theevolutionary relationships of one genetic sequence to another that isgenerated using a defined set of phylogenetic reconstruction algorithms(e.g. parsimony, maximum likelihood, or Bayesian). Nodes in the treerepresent distinct ancestral sequences and the confidence of any node isprovided by a bootstrap or Bayesian posterior probability, whichmeasures branch uncertainty.

“Operational taxonomic unit (OTU, plural OTUs)” refers to a terminalleaf in a phylogenetic tree and is defined by a specific geneticsequence and all sequences that share sequence identity to this sequenceat the level of species. A “type” or a plurality of “types” of bacteriaincludes an OTU or a plurality of different OTUs, and also encompasses astrain, species, genus, family or order of bacteria. The specificgenetic sequence may be the 16S sequence or a portion of the 16Ssequence or it may be a functionally conserved housekeeping gene foundbroadly across the eubacterial kingdom. OTUs share at least 95%, 96%,97%, 98%, or 99% sequence identity. OTUs are frequently defined bycomparing sequences between organisms. Sequences with less than 95%sequence identity are not considered to form part of the same OTU.

“Clade” refers to the set of OTUs or members of a phylogenetic treedownstream of a statistically valid node in a phylogenetic tree. Theclade comprises a set of terminal leaves in the phylogenetic tree thatis a distinct monophyletic evolutionary unit.

In microbiology, “16S sequencing” or “16S rRNA” or “16S-rRNA” or “16S”refers to sequence derived by characterizing the nucleotides thatcomprise the 16S ribosomal RNA gene(s). The bacterial 16S rDNA isapproximately 1500 nucleotides in length and is used in reconstructingthe evolutionary relationships and sequence similarity of one bacterialisolate to another using phylogenetic approaches. 16S sequences are usedfor phylogenetic reconstruction as they are in general highly conserved,but contain specific hypervariable regions that harbor sufficientnucleotide diversity to differentiate genera and species of mostbacteria, as well as fungi.

The “V1-V9 regions” of the 16S rRNA refers to the first through ninthhypervariable regions of the 16S rRNA gene that are used for genetictyping of bacterial samples. These regions in bacteria are defined bynucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043,1117-1173, 1243-1294 and 1435-1465 respectively using numbering based onthe E. coli system of nomenclature. Brosius et al., Complete nucleotidesequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS75(10):4801-4805 (1978). In some embodiments, at least one of the V1,V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize anOTU. In one embodiment, the V1, V2, and V3 regions are used tocharacterize an OTU. In another embodiment, the V3, V4, and V5 regionsare used to characterize an OTU. In another embodiment, the V4 region isused to characterize an OTU. A person of ordinary skill in the art canidentify the specific hypervariable regions of a candidate 16S rRNA (inSEQ ID NOs 1-1,864) by comparing the candidate sequence in question tothe reference sequence and identifying the hypervariable regions basedon similarity to the reference hypervariable regions.

The terms “subject” or “patient” refers to any animal subject includinghumans, laboratory animals (e.g., primates, rats, mice), livestock(e.g., cows, sheep, goats, pigs, turkeys, chickens), and household pets(e.g., dogs, cats, rodents, etc.). The subject or patient may behealthy, or may be suffering from an infection due to a gastrointestinalpathogen or may be at risk of developing or transmitting to others aninfection due to a gastrointestinal pathogen.

The term “pathobiont” refer to specific bacterial species found inhealthy hosts that may trigger immune-mediated pathology and/or diseasein response to certain genetic or environmental factors. Chow et al.,(2011) Curr Op Immunol. Pathobionts of the intestinal microbiota andinflammatory disease. 23: 473-80. Thus, a pathobiont is a pathogen thatis mechanistically distinct from an acquired infectious organism. Thus,the term “pathogen” includes both acquired infectious organisms andpathobionts.

DETAILED DESCRIPTION

Bacterial Compositions

Provided are bacteria and combinations of bacteria of the human gutmicrobiota with the capacity to meaningfully provide functions of ahealthy microbiota or catalyze an augmentation to the residentmicrobiome when administered to mammalian hosts. In particular, providedare synergistic combinations that treat, prevent, delay or reduce thesymptoms of diseases, disorders and conditions associated with adysbiosis. Representative diseases, disorders and conditions potentiallyassociated with a dysbiosis, which are suitable for treatment with thecompositions and methods as described herein, are provided in Tables 8and 9. Without being limited to a specific mechanism, it is thought thatsuch compositions inhibit the growth, proliferation, and/or colonizationof one or a plurality of pathogenic bacteria in the dysbioticmicrobiotal niche, so that a healthy, diverse and protective microbiotacolonizes and populates the intestinal lumen to establish or reestablishecological control over pathogens or potential pathogens (e.g., somebacteria are pathogenic bacteria only when present in a dysbioticenvironment). Inhibition of pathogens includes those pathogens such asC. difficile, Salmonella spp., enteropathogenic E coli, multi-drugresistant bacteria such as Klebsiella, and E. coli, Carbapenem-resistentEnterobacteriaceae (CRE), extended spectrum beta-lactam resistantEnterococci (ESBL), and vancomycin-resistant Enterococci (VRE).

The bacterial compositions provided herein are produced and the efficacythereof in inhibiting pathogenic bacteria is demonstrated as provided infurther detail herein.

In particular, in order to characterize those antagonistic relationshipsbetween gut commensals that are relevant to the dynamics of themammalian gut habitat, provided is an in vitro microplate-basedscreening system that demonstrates the efficacy of those bacterialcompositions, including the ability to inhibit (or antagonize) thegrowth of a bacterial pathogen or pathobiont, typically agastrointestinal microorganism. These methods provide novel combinationsof gut microbiota species and OTUs that are able to restore or enhanceecological control over important gut pathogens or pathobionts in vivo.

Bacterial compositions may comprise two types of bacteria (termed“binary combinations” or “binary pairs”) or greater than two types ofbacteria. Bacterial compositions that comprise three types of bacteriaare termed “ternary combinations”. For instance, a bacterial compositionmay comprise at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, or at least 21, 22, 23, 24, 25, 26,27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or at least 40, atleast 50 or greater than 50 types of bacteria, as defined by species oroperational taxonomic unit (OTU), or otherwise as provided herein. Inone embodiment, the composition comprises at least two types of bacteriachosen from Table 1.

In another embodiment, the number of types of bacteria present in abacterial composition is at or below a known value. For example, in suchembodiments the bacterial composition comprises 50 or fewer types ofbacteria, such as 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer, or 9 or fewer types ofbacteria, 8 or fewer types of bacteria, 7 or fewer types of bacteria, 6or fewer types of bacteria, 5 or fewer types of bacteria, 4 or fewertypes of bacteria, or 3 or fewer types of bacteria. In anotherembodiment, a bacterial composition comprises from 2 to no more than 40,from 2 to no more than 30, from 2 to no more than 20, from 2 to no morethan 15, from 2 to no more than 10, or from 2 to no more than 5 types ofbacteria.

In some embodiments, bacterial compositions are provided with theability to exclude pathogenic bacteria. Exemplary bacterial compositionsare demonstrated to reduce the growth rate of one pathogen, C.difficile, as provided in the Examples, wherein the ability of thebacterial compositions is demonstrated by assessing the antagonismactivity of a combination of OTUs or strains towards a given pathogenusing in vitro assays.

In some embodiments, bacterial compositions with the capacity to durablyexclude C. difficile, are developed using a methodology for estimatingan Ecological Control Factor (ECF) for constituents within the humanmicrobiota. The ECF is determined by assessing the antagonistic activityof a given commensal strain or combination of strains towards a givenpathogen using an in vitro assay, resulting in observed levels ofecological control at various concentrations of the added commensalstrains. The ECF for a commensal strain or combination of strains issomewhat analogous to the longstanding minimal inhibitory concentration(MIC) assessment that is employed in the assessment of antibiotics. TheECF allows for the assessment and ranking of relative potencies ofcommensal strains and combinations of strains for their ability toantagonize gastrointestinal pathogens. The ECF of a commensal strain orcombination of strains may be calculated by assessing the concentrationof that composition that is able to mediate a given percentage ofinhibition (e.g., at least 10%, 20%, 50%, 70%, 75%, 80%, 85%, 90%, 95%,or 100%) of a target pathogen in the in vitro assay. Provided herein arecombinations of strains or OTUs within the human microbiota that areable to significantly reduce the rate of gastrointestinal pathogenreplication within the in vitro assay. These compositions are capable ofproviding a safe and effective means by which to affect the growth,replication, and disease severity of such bacterial pathogens.

Bacterial compositions may be prepared comprising at least two types ofisolated bacteria, wherein a first type and a second type areindependently chosen from the species or OTUs listed in Table 1 and SEQID NOs. 1-1,864. Certain embodiments of bacterial compositions with atleast two types of isolated bacteria containing binary pairs arereflected in Table 2. Additionally, a bacterial composition may beprepared comprising at least two types of isolated bacteria, wherein afirst OTU and a second OTU are independently characterized by, i.e., atleast 95%, 96%, 97%, 98%, 99% or including 100% sequence identity to,sequences listed in SEQ ID NOs. 1-1,864. Generally, the first bacteriaand the second bacteria are not the same OTU. The sequences provided inSEQ ID NOs. 1-1,864 are full 16S sequences. Therefore, in oneembodiment, the first and/or second OTUs may be characterized by thefull 16S sequences listed in SEQ ID NOs. 1-1,864. In another embodiment,the first and/or second OTUs may be characterized by one or more of thevariable regions of the 16S sequence (V1-V9). These regions in bacteriaare defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879,986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature. (See, e.g.,Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA genefrom Escherichia coli, PNAS 75(10):4801-4805 (1978)). In someembodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9regions are used to characterize an OTU. In one embodiment, the V1, V2,and V3 regions are used to characterize an OTU. In another embodiment,the V3, V4, and V5 regions are used to characterize an OTU. In anotherembodiment, the V4 region is used to characterize an OTU.

Methods for Determining 16S Sequence.

OTUs may be defined either by full 16S sequencing of the rRNA gene, bysequencing of a specific hypervariable region of this gene (i.e. V1, V2,V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination ofhypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial16S rDNA is approximately 1500 nucleotides in length and is used inreconstructing the evolutionary relationships and sequence similarity ofone bacterial isolate to another using phylogenetic approaches. 16Ssequences are used for phylogenetic reconstruction as they are ingeneral highly conserved, but contain specific hypervariable regionsthat harbor sufficient nucleotide diversity to differentiate genera andspecies of most microbes. Using well known techniques, in order todetermine the full 16S sequence or the sequence of any hypervariableregion of the 16S sequence, genomic DNA is extracted from a bacterialsample, the 16S rDNA (full region or specific hypervariable regions)amplified using polymerase chain reaction (PCR), the PCR productscleaned, and nucleotide sequences delineated to determine the geneticcomposition of 16S gene or subdomain of the gene. If full 16S sequencingis performed, the sequencing method used may be, but is not limited to,Sanger sequencing. If one or more hypervariable regions are used, suchas the V4 region, the sequencing may be, but is not limited to being,performed using the Sanger method or using a next-generation sequencingmethod, such as an Illumina (sequencing by synthesis) method usingbarcoded primers allowing for multiplex reactions.

Bacterial Compositions Exclusive of Certain Bacterial Species orStrains.

In one embodiment, the bacterial composition does not comprise at leastone of Enterococcus faecalis (previously known as Streptococcusfaecalis), Clostridium innocuum, Clostridium ramosum, Bacteroidesovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichiacoli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta(previously known as Peptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise atleast one of Acidaminococcus intestinalis, Bacteroides ovatus, twospecies of Bifidobacterium adolescentis, two species of Bifidobacteriumlongum, Collinsella aerofaciens, two species of Dorea longicatena,Escherichia coli, Eubacterium eligens, Eubacterium limosum, four speciesof Eubacterium rectale, Eubacterium ventriosumi, Faecalibacteriumprausnitzii, Lactobacillus casei, Lactobacillus paracasei,Paracateroides distasonis, Raoultella sp., one species of Roseburia(chosen from Roseburia faecalis or Roseburia faecis), Roseburiaintestinalis, two species of Ruminococcus torques, and Streptococcusmitis.

In another embodiment, the bacterial composition does not comprise atleast one of Barnesiella intestinihominis; Lactobacillus reuteri; aspecies characterized as one of Enterococcus hirae, Enterococus faecium,or Enterococcus durans; a species characterized as one of Anaerostipescaccae or Clostridium indolis; a species characterized as one ofStaphylococcus warneri or Staphylococcus pasteuri; and Adlercreutziaequolfaciens.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium absonum, Clostridium argentinense, Clostridiumbaratii, Clostridium bifermentans, Clostridium botulinum, Clostridiumbutyricum, Clostridium cadaveris, Clostridium camis, Clostridiumcelatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium difficile, Clostridium fallax, Clostridiumfelsineum, Clostridium ghonii, Clostridium glycolicum, Clostridiumhaemolyticum, Clostridium hastiforme, Clostridium histolyticum,Clostridium indolis, Clostridium innocuum, Clostridium irregulare,Clostridium limosum, Clostridium malenominatum, Clostridium novyi,Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense,Clostridium sartagoforme, Clostridium scindens, Clostridium septicum,Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum,Clostridium tertium, Clostridium tetani, Clostridium welchii, andClostridium villosum.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium innocuum, Clostridum bifermentans, Clostridiumbutyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron,Bacteroides uniformis, three strains of Escherichia coli, andLactobacillus sp.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium bifermentans, Clostridium innocuum, Clostridiumbutyricum, three strains of Escherichia coli, three strains ofBacteroides, and Blautia producta (previously known asPeptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides sp., Escherichia coli, and non pathogenicClostridia, including Clostridium innocuum, Clostridium bifermentans andClostridium ramosum.

In another embodiment, the bacterial composition does not comprise atleast one of more than one Bacteroides species, Escherichia coli andnon-pathogenic Clostridia, such as Clostridium butyricum, Clostridiumbifermentans and Clostridium innocuum.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides caccae, Bacteroides capillosus, Bacteroidescoagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroidesforsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroidesgracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae,Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis,Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris,Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides ureolyticus, and Bacteroides vulgatus.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides, Eubacteria, Fusobacteria, Propionibacteria,Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger,Desulfomonas, and Peptostreptococcus.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens,Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previouslyknown as Peptostreptococcus productus II), Bacteroides fragilis ss.Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacteriumaerofaciens III, Blautia producta (previously known asPeptostreptococcus productus I), Ruminococcus bromii, Bifidobacteriumadolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacteriumsiraeum, Ruminococcus torques, Eubacterium rectale III-H, Eubacteriumrectale IV, Eubacterium eligens, Bacteroides eggerthii, Clostridiumleptum, Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacteriuminfantis, Eubacterium rectale III-F, Coprococcus comes, Bacteroidescapillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacteriumhali, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcusobeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillusleichmanii, Ruminococcus cailidus, Butyrivibrio crossotus,Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilisss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum,Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1,Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum,Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferum I,Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum,Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT,Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroidesfragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacteriumnucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcusmorbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcusintermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X,Coprococcus BH, -CC; Eubacterium tenue, Eubacterium ramulus, EubacteriumAE, -AG-H, -AG-M, -AJ, -BN-1; Bacteroides clostridiiformis ss.clostridformis, Bacteroides coagulans, Bacteroides orails, Bacteroidesruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus,Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H,Lactobacillus G, and Succinivibrio A.

Inhibition of Bacterial Pathogens.

The bacterial compositions offer a protective or therapeutic effectagainst infection by one or more GI pathogens of interest.

A list of exemplary bacterial pathogens is provided in Table 5.

In some embodiments, the pathogenic bacterium is selected from the groupconsisting of Yersinia, Vibrio, Treponema, Streptococcus,Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas,Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella,Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia,Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium,Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella,Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug resistantbacteria, extended spectrum beta-lactam resistant Enterococci (ESBL),Carbapenem-resistent Enterobacteriaceae (CRE), and vancomycin-resistantEnterococci (VRE).

In some embodiments, these pathogens include, but are not limited to,Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides,Bacillus cereus, Campylobacter jejuni, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, enteroaggregativeEscherichia coli, enterohemorrhagic Escherichia coli, enteroinvasiveEscherichia coli, enterotoxigenic Escherichia coli (such as, but notlimited to, LT and/or ST), Escherichia coli 0157:H7, Helicobacterpylori, Klebsiellia pneumonia, Lysteria monocytogenes, Plesiomonasshigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi,Shigella spp., Staphylococcus spp., Staphylococcus aureus,vancomycin-resistant enterococcus spp., Vibrio spp., Vibrio cholerae,Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica.

In one embodiment, the pathogen of interest is at least one pathogenchosen from Clostridium difficile, Salmonella spp., pathogenicEscherichia coli, vancomycin-resistant Enterococcus spp., and extendedspectrum beta-lactam resistant Enterococci (ESBL).

In Vitro Assays Substantiating Protective Effect of BacterialCompositions.

In one embodiment, provided is an In Vitro Assay utilizing competitionbetween the bacterial compositions or subsets thereof and C. difficile.Exemplary embodiments of this Assay are provided herein and in theExamples.

In another embodiment, provided is an In Vitro Assay utilizing 10%(wt/vol) Sterile-Filtered Feces. Provided is an in vitro assay to testfor the protective effect of the bacterial compositions and to screen invitro for combinations of microbes that inhibit the growth of apathogen. The assay can operate in automated high-throughput or manualmodes. Under either system, human or animal feces may be re-suspended inan anaerobic buffer solution, such as pre-reduced PBS or other suitablebuffer, the particulate removed by centrifugation, and filtersterilized. This 10% sterile-filtered feces material serves as the basemedia for the in vitro assay. To test a bacterial composition, aninvestigator may add it to the sterile-filtered feces material for afirst incubation period and then may inoculate the incubated microbialsolution with the pathogen of interest for a second incubation period.The resulting titer of the pathogen may be quantified by any number ofmethods such as those described below, and the change in the amount ofpathogen is compared to standard controls including the pathogencultivated in the absence of the bacterial composition. The assay isconducted using at least one control. Feces from a healthy subject maybe used as a positive control. As a negative control, antibiotic-treatedfeces or heat-treated feces may be used. Various bacterial compositionsmay be tested in this material and the bacterial compositions optionallycompared to the positive and/or negative controls. The ability toinhibit the growth of the pathogen may be measured by plating theincubated material on C. difficile selective media and countingcolonies. After competition between the bacterial composition and C.difficile, each well of the in vitro assay plate is serially dilutedten-fold six times, and plated on selective media, such as but notlimited to cycloserine cefoxitin mannitol agar (CCMA) or cycloserinecefoxitin fructose agar (CCFA), and incubated. Colonies of C. difficileare then counted to calculate the concentration of viable cells in eachwell at the end of the competition. Colonies of C. difficile areconfirmed by their characteristic diffuse colony edge morphology as wellas fluorescence under UV light.

In another embodiment, the in vitro assay utilizes Antibiotic-TreatedFeces. In an alternative embodiment, and instead of using 10%sterile-filtered feces, human or animal feces may be resuspended in ananaerobic buffer solution, such as pre-reduced PBS or other suitablebuffer. The resuspended feces is treated with an antibiotic, such asclindamycin, or a cocktail of several antibiotics in order to reduce theability of feces from a healthy subject to inhibit the growth of C.difficile; this material is termed the antibiotic-treated matrix. Whilenot being bound by any mechanism, it is believed that beneficialbacteria in healthy subjects protects them from infection by competingout C. difficile. Treating feces with antibiotics kills or reduces thepopulation of those beneficial bacteria, allowing C. difficile to growin this assay matrix. Antibiotics in addition to clindamycin thatinhibit the normal flora include ceftriaxone and piperacillin-tazobactamand may be substituted for the clindamycin. The antibiotic-treatedmatrix is centrifuged, the supernatant removed, and the pelletedmaterial resuspended in filter-sterilized, diluted feces in order toremove any residual antibiotic. This washed antibiotic-treated matrixmay be used in the in vitro assay described above in lieu of the 10%sterile-filtered feces.

Alternatively, the ability to inhibit the growth of the pathogen may bemeasured by quantitative PCR (qPCR). Standard techniques may be followedto generate a standard curve for the pathogen of interest. Genomic DNAmay be extracted from samples using commercially-available kits, such asthe Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit(Mo Bio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool MiniKit (QIAGEN, Valencia, Calif.) according to the manufacturer'sinstructions. The qPCR may be conducted using HotMasterMix (5PRIME,Gaithersburg, Md.) and primers specific for the pathogen of interest,and may be conducted on a MicroAmp® Fast Optical 96-well Reaction Platewith Barcode (0.1 mL) (Life Technologies, Grand Island, N.Y.) andperformed on a BioRad C1000™ Thermal Cycler equipped with a CFX96™Real-Time System (BioRad, Hercules, Calif.), with fluorescent readingsof the FAM and ROX channels. The Cq value for each well on the FAMchannel is determined by the CFX Manager™ software version 2.1. Thelog₁₀ (cfu/ml) of each experimental sample is calculated by inputting agiven sample's Cq value into linear regression model generated from thestandard curve comparing the Cq values of the standard curve wells tothe known log₁₀ (cfu/ml) of those samples. The skilled artisan mayemploy alternative qPCR modes.

Also provided are In Vivo Assay Establishing Protective Effect ofBacterial Compositions. Provided is an in vivo mouse model to test forthe protective effect of the bacterial compositions against C.difficile. In this model (based on Chen, et al., A mouse model ofClostridium difficile associated disease, Gastroenterology135(6):1984-1992 (2008)), mice are made susceptible to C. difficile by a7 day treatment (days −12 to −5 of experiment) with 5 to 7 antibiotics(including kanamycin, colistin, gentamycin, metronidazole and vancomycinand optionally including ampicillin and ciprofloxacin) delivered viatheir drinking water, followed by a single dose with Clindamycin on day−3, then challenged three days later on day 0 with 10⁴ spores of C.difficile via oral gavage (i.e., oro-gastric lavage). Bacterialcompositions may be given either before (prophylactic treatment) orafter (therapeutic treatment) C. difficile gavage. Further, bacterialcompositions may be given after (optional) vancomycin treatment (seebelow) to assess their ability to prevent recurrence and thus suppressthe pathogen in vivo. The outcomes assessed each day from day −1 to day6 (or beyond, for prevention of recurrence) are weight, clinical signs,mortality and shedding of C. difficile in the feces. Weight loss,clinical signs of disease, and C. difficile shedding are typicallyobserved without treatment. Vancomycin provided by oral gavage on days−1 to 4 protects against these outcomes and serves as a positivecontrol. Clinical signs are subjective, and scored each day by the sameexperienced observer. Animals that lose greater than or equal to 25% oftheir body weight are euthanized and counted as infection-relatedmortalities. Feces are gathered from mouse cages (5 mice per cage) eachday, and the shedding of C. difficile spores is detected in the fecesusing a selective plating assay as described for the in vitro assayabove, or via qPCR for the toxin gene as described herein. The effectsof test materials including 10% suspension of human feces (as a positivecontrol), bacterial compositions, or PBS (as a negative vehiclecontrol), are determined by introducing the test article in a 0.2 mLvolume into the mice via oral gavage on day −1, one day prior to C.difficile challenge, on day 1, 2 and 3 as treatment or post-vancomycintreatment on days 5, 6, 7 and 8. Vancomycin, as discussed above, isgiven on days 1 to 4 as another positive control. Alternative dosingschedules and routes of administration (e.g. rectal) may be employed,including multiple doses of test article, and 10³ to 10¹⁰ of a givenorganism or composition may be delivered.

Methods for Preparing a Bacterial Composition for Administration to aSubject.

Methods for producing bacterial compositions may include three mainprocessing steps, combined with one or more mixing steps. The steps are:organism banking, organism production, and preservation.

For banking, the strains included in the bacterial composition may be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments using a culturing step, the agar or broth may containnutrients that provide essential elements and specific factors thatenable growth. An example would be a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1mg/L menadione. A variety of microbiological media and variations arewell known in the art (e.g. R. M. Atlas, Handbook of MicrobiologicalMedia (2010) CRC Press). Medium can be added to the culture at thestart, may be added during the culture, or may beintermittently/continuously flowed through the culture. The strains inthe bacterial composition may be cultivated alone, as a subset of thebacterial composition, or as an entire collection comprising thebacterial composition. As an example, a first strain may be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for atime sufficient to build biomass. For bacterial compositions for humanuse this is often at 37° C. temperature, pH, and other parameter withvalues similar to the normal human niche. The environment may beactively controlled, passively controlled (e.g., via buffers), orallowed to drift. For example, for anaerobic bacterial compositions(e.g., gut microbiota), an anoxic/reducing environment may be employed.This can be accomplished by addition of reducing agents such as cysteineto the broth, and/or stripping it of oxygen. As an example, a culture ofa bacterial composition may be grown at 37° C., pH 7, in the mediumabove, pre-reduced with 1 g/L cysteine D HCl.

When the culture has generated sufficient biomass, it may be preservedfor banking. The organisms may be placed into a chemical milieu thatprotects from freezing (adding ‘cryoprotectants’), drying(‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then treating the culture for preservation. Containers aregenerally impermeable and have closures that assure isolation from theenvironment. Cryopreservation treatment is accomplished by freezing aliquid at ultra-low temperatures (e.g., at or below −80° C.). Driedpreservation removes water from the culture by evaporation (in the caseof spray drying or ‘cool drying’) or by sublimation (e.g., for freezedrying, spray freeze drying). Removal of water improves long-termbacterial composition storage stability at temperatures elevated abovecryogenic. If the bacterial composition comprises spore forming speciesand results in the production of spores, the final composition may bepurified by additional means such as density gradient centrifugationpreserved using the techniques described above. Bacterial compositionbanking may be done by culturing and preserving the strainsindividually, or by mixing the strains together to create a combinedbank. As an example of cryopreservation, a bacterial composition culturemay be harvested by centrifugation to pellet the cells from the culturemedium, the supernate decanted and replaced with fresh culture brothcontaining 15% glycerol. The culture can then be aliquoted into 1 mLcryotubes, sealed, and placed at −80° C. for long-term viabilityretention. This procedure achieves acceptable viability upon recoveryfrom frozen storage.

Organism production may be conducted using similar culture steps tobanking, including medium composition and culture conditions. It may beconducted at larger scales of operation, especially for clinicaldevelopment or commercial production. At larger scales, there may beseveral subcultivations of the bacterial composition prior to the finalcultivation. At the end of cultivation, the culture is harvested toenable further formulation into a dosage form for administration. Thiscan involve concentration, removal of undesirable medium components,and/or introduction into a chemical milieu that preserves the bacterialcomposition and renders it acceptable for administration via the chosenroute. For example, a bacterial composition may be cultivated to aconcentration of 10¹⁰ CFU/mL, then concentrated 20-fold by tangentialflow microfiltration; the spent medium may be exchanged by diafilteringwith a preservative medium consisting of 2% gelatin, 100 mM trehalose,and 10 mM sodium phosphate buffer. The suspension can then befreeze-dried to a powder and titrated.

After drying, the powder may be blended to an appropriate potency, andmixed with other cultures and/or a filler such as microcrystallinecellulose for consistency and ease of handling, and the bacterialcomposition formulated as provided herein.

Formulations.

Provided are formulations for administration to humans and othersubjects in need thereof. Generally the bacterial compositions arecombined with additional active and/or inactive materials in order toproduce a final product, which may be in single dosage unit or in amulti-dose format.

In some embodiments the composition comprises at least one carbohydrate.A “carbohydrate” refers to a sugar or polymer of sugars. The terms“saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide”may be used interchangeably. Most carbohydrates are aldehydes or ketoneswith many hydroxyl groups, usually one on each carbon atom of themolecule. Carbohydrates generally have the molecular formulaC_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments the composition comprises at least one lipid. Asused herein a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In some embodiments thecomposition comprises at least one modified lipid, for example a lipidthat has been modified by cooking.

In some embodiments the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments the composition comprises at least one supplementalvitamin. The at least one vitamin can be fat-soluble or water solublevitamins. Suitable vitamins include but are not limited to vitamin C,vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin,vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenicacid, and biotin. Suitable forms of any of the foregoing are salts ofthe vitamin, derivatives of the vitamin, compounds having the same orsimilar activity of the vitamin, and metabolites of the vitamin.

In some embodiments the composition comprises an excipient. Non-limitingexamples of suitable excipients include a buffering agent, apreservative, a stabilizer, a binder, a compaction agent, a lubricant, adispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In some embodiments the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments the excipient comprises a preservative. Non-limitingexamples of suitable preservatives include antioxidants, such asalpha-tocopherol and ascorbate, and antimicrobials, such as parabens,chlorobutanol, and phenol.

In some embodiments the composition comprises a binder as an excipient.Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In some embodiments the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In some embodiments the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments the composition comprises a disintegrant as anexcipient. In some embodiments the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, micro-crystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pecitin, andtragacanth. In some embodiments the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments the excipient comprises a flavoring agent. Flavoringagents can be chosen from synthetic flavor oils and flavoring aromatics;natural oils; extracts from plants, leaves, flowers, and fruits; andcombinations thereof. In some embodiments the flavoring agent isselected from cinnamon oils; oil of wintergreen; peppermint oils; cloveroil; hay oil; anise oil; eucalyptus; vanilla; citrus oil such as lemonoil, orange oil, grape and grapefruit oil; and fruit essences includingapple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, andapricot.

In some embodiments the excipient comprises a sweetener. Non-limitingexamples of suitable sweeteners include glucose (corn syrup), dextrose,invert sugar, fructose, and mixtures thereof (when not used as acarrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; and sugar alcohols such as sorbitol,mannitol, sylitol, and the like. Also contemplated are hydrogenatedstarch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In some embodiments the composition comprises a coloring agent.Non-limiting examples of suitable color agents include food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), and externaldrug and cosmetic colors (Ext. D&C). The coloring agents can be used asdyes or their corresponding lakes.

The weight fraction of the excipient or combination of excipients in theformulation is usually about 99% or less, such as about 95% or less,about 90% or less, about 85% or less, about 80% or less, about 75% orless, about 70% or less, about 65% or less, about 60% or less, about 55%or less, 50% or less, about 45% or less, about 40% or less, about 35% orless, about 30% or less, about 25% or less, about 20% or less, about 15%or less, about 10% or less, about 5% or less, about 2% or less, or about1% or less of the total weight of the composition.

The bacterial compositions disclosed herein can be formulated into avariety of forms and administered by a number of different means. Thecompositions can be administered orally, rectally, or parenterally, informulations containing conventionally acceptable carriers, adjuvants,and vehicles as desired. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, or intrasternal injection andinfusion techniques. In an exemplary embodiment, the bacterialcomposition is administered orally.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, troches, lozenges, powders, and granules. A capsuletypically comprises a core material comprising a bacterial compositionand a shell wall that encapsulates the core material. In someembodiments the core material comprises at least one of a solid, aliquid, and an emulsion. In some embodiments the shell wall materialcomprises at least one of a soft gelatin, a hard gelatin, and a polymer.Suitable polymers include, but are not limited to: cellulosic polymerssuch as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose (HPMC), methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulosesuccinate and carboxymethylcellulose sodium; acrylic acid polymers andcopolymers, such as those formed from acrylic acid, methacrylic acid,methyl acrylate, ammonio methylacrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate (e.g., those copolymers soldunder the trade name “Eudragit”); vinyl polymers and copolymers such aspolyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate,vinylacetate crotonic acid copolymer, and ethylene-vinyl acetatecopolymers; and shellac (purified lac). In some embodiments at least onepolymer functions as taste-masking agents.

Tablets, pills, and the like can be compressed, multiply compressed,multiply layered, and/or coated. The coating can be single or multiple.In one embodiment, the coating material comprises at least one of asaccharide, a polysaccharide, and glycoproteins extracted from at leastone of a plant, a fungus, and a microbe. Non-limiting examples includecorn starch, wheat starch, potato starch, tapioca starch, cellulose,hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin,mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gumkaraya, gum ghatti, tragacanth gum, funori, carrageenans, agar,alginates, chitosans, or gellan gum. In some embodiments the coatingmaterial comprises a protein. In some embodiments the coating materialcomprises at least one of a fat and an oil. In some embodiments the atleast one of a fat and an oil is high temperature melting. In someembodiments the at least one of a fat and an oil is hydrogenated orpartially hydrogenated. In some embodiments the at least one of a fatand an oil is derived from a plant. In some embodiments the at least oneof a fat and an oil comprises at least one of glycerides, free fattyacids, and fatty acid esters. In some embodiments the coating materialcomprises at least one edible wax. The edible wax can be derived fromanimals, insects, or plants. Non-limiting examples include beeswax,lanolin, bayberry wax, carnauba wax, and rice bran wax. Tablets andpills can additionally be prepared with enteric coatings.

Alternatively, powders or granules embodying the bacterial compositionsdisclosed herein can be incorporated into a food product. In someembodiments the food product is a drink for oral administration.Non-limiting examples of a suitable drink include fruit juice, a fruitdrink, an artificially flavored drink, an artificially sweetened drink,a carbonated beverage, a sports drink, a liquid diary product, a shake,an alcoholic beverage, a caffeinated beverage, infant formula and soforth. Other suitable means for oral administration include aqueous andnonaqueous solutions, emulsions, suspensions and solutions and/orsuspensions reconstituted from non-effervescent granules, containing atleast one of suitable solvents, preservatives, emulsifying agents,suspending agents, diluents, sweeteners, coloring agents, and flavoringagents.

In some embodiments the food product is a solid foodstuff. Suitableexamples of a solid foodstuff include without limitation a food bar, asnack bar, a cookie, a brownie, a muffin, a cracker, an ice cream bar, afrozen yogurt bar, and the like.

In some embodiments, the compositions disclosed herein are incorporatedinto a therapeutic food. In some embodiments, the therapeutic food is aready-to-use food that optionally contains some or all essentialmacronutrients and micronutrients. In some embodiments, the compositionsdisclosed herein are incorporated into a supplementary food that isdesigned to be blended into an existing meal. In some embodiments, thesupplemental food contains some or all essential macronutrients andmicronutrients. In some embodiments, the bacterial compositionsdisclosed herein are blended with or added to an existing food tofortify the food's protein nutrition. Examples include food staples(grain, salt, sugar, cooking oil, margarine), beverages (coffee, tea,soda, beer, liquor, sports drinks), snacks, sweets and other foods.

In one embodiment, the formulations are filled into gelatin capsules fororal administration. An example of an appropriate capsule is a 250 mggelatin capsule containing from 10 (up to 100 mg) of lyophilized powder(10⁸ to 10¹¹ bacteria), 160 mg microcrystalline cellulose, 77.5 mggelatin, and 2.5 mg magnesium stearate. In an alternative embodiment,from 10⁵ to 10¹² bacteria may be used, 10⁵ to 10⁷, 10 ⁶ to 10⁷, or 10⁸to 10¹⁰, with attendant adjustments of the excipients if necessary. Inan alternative embodiment an enteric-coated capsule or tablet or with abuffering or protective composition may be used.

In one embodiment, the number of bacteria of each type may be present inthe same amount or in different amounts. For example, in a bacterialcomposition with two types of bacteria, the bacteria may be present infrom a 1:10,000 ratio to a 1:1 ratio, from a 1:10,000 ratio to a 1:1,000ratio, from a 1:1,000 ratio to a 1:100 ratio, from a 1:100 ratio to a1:50 ratio, from a 1:50 ratio to a 1:20 ratio, from a 1:20 ratio to a1:10 ratio, from a 1:10 ratio to a 1:1 ratio. For bacterial compositionscomprising at least three types of bacteria, the ratio of type ofbacteria may be chosen pairwise from ratios for bacterial compositionswith two types of bacteria. For example, in a bacterial compositioncomprising bacteria A, B, and C, at least one of the ratio betweenbacteria A and B, the ratio between bacteria B and C, and the ratiobetween bacteria A and C may be chosen, independently, from the pairwisecombinations above.

Methods of Treating a Subject.

In some embodiments the proteins and compositions disclosed herein areadministered to a patient or a user (sometimes collectively referred toas a “subject”). As used herein “administer” and “administration”encompasses embodiments in which one person directs another to consume abacterial composition in a certain manner and/or for a certain purpose,and also situations in which a user uses a bacteria composition in acertain manner and/or for a certain purpose independently of or invariance to any instructions received from a second person. Non-limitingexamples of embodiments in which one person directs another to consume abacterial composition in a certain manner and/or for a certain purposeinclude when a physician prescribes a course of conduct and/or treatmentto a patient, when a parent commands a minor user (such as a child) toconsume a bacterial composition, when a trainer advises a user (such asan athlete) to follow a particular course of conduct and/or treatment,and when a manufacturer, distributer, or marketer recommends conditionsof use to an end user, for example through advertisements or labeling onpackaging or on other materials provided in association with the sale ormarketing of a product.

The bacterial compositions offer a protective and/or therapeutic effectagainst infection by one or more GI pathogens of interest and thus maybe administered after an acute case of infection has been resolved inorder to prevent relapse, during an acute case of infection as acomplement to antibiotic therapy if the bacterial composition is notsensitive to the same antibiotics as the GI pathogen, or to preventinfection or reduce transmission from disease carriers. These pathogensinclude, but are not limited to, Aeromonas hydrophila, Campylobacterfetus, Plesiomonas shigelloides, Bacillus cereus, Campylobacter jejuni,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli,enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli (LTand/or ST), Escherichia coli 0157:H7, Helicobacter pylori, Klebsiellapneumonia, Lysteria monocytogenes, Plesiomonas shigelloides, Salmonellaspp., Salmonella typhi, Shigella spp., Staphylococcus, Staphylococcusaureus, vancomycin-resistant Enterococcus spp., Vibrio spp., Vibriocholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersiniaenterocolitica.

In one embodiment, the pathogen may be Clostridium difficile, Salmonellaspp., pathogenic Escherichia coli, Carbapenem-resistentEnterobacteriaceae (CRE), extended spectrum beta-lactam resistantEnterococci (ESBL) and vancomycin-resistant Enterococci (VRE). In yetanother embodiment, the pathogen may be Clostridium difficile.

The present bacterial compositions may be useful in a variety ofclinical situations. For example, the bacterial compositions may beadministered as a complementary treatment to antibiotics when a patientis suffering from an acute infection, to reduce the risk of recurrenceafter an acute infection has subsided, or when a patient will be inclose proximity to others with or at risk of serious gastrointestinalinfections (physicians, nurses, hospital workers, family members ofthose who are ill or hospitalized).

The present bacterial compositions may be administered to animals,including humans, laboratory animals (e.g., primates, rats, mice),livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), andhousehold pets (e.g., dogs, cats, rodents).

In the present method, the bacterial composition is administeredenterically, in other words by a route of access to the gastrointestinaltract. This includes oral administration, rectal administration(including enema, suppository, or colonoscopy), by an oral or nasal tube(nasogastric, nasojejunal, oral gastric, or oral jejunal), as detailedmore fully herein.

A. Pretreatment Protocols

Prior to administration of the bacterial composition, the patient mayoptionally have a pretreatment protocol to prepare the gastrointestinaltract to receive the bacterial composition. In certain embodiments, thepretreatment protocol is advisable, such as when a patient has an acuteinfection with a highly resilient pathogen. In other embodiments, thepretreatment protocol is entirely optional, such as when the pathogencausing the infection is not resilient, or the patient has had an acuteinfection that has been successfully treated but where the physician isconcerned that the infection may recur. In these instances, thepretreatment protocol may enhance the ability of the bacterialcomposition to affect the patient's microbiome.

As one way of preparing the patient for administration of the microbialecosystem, at least one antibiotic may be administered to alter thebacteria in the patient. As another way of preparing the patient foradministration of the microbial ecosystem, a standard colon-cleansingpreparation may be administered to the patient to substantially emptythe contents of the colon, such as used to prepare a patient for acolonscopy. By “substantially emptying the contents of the colon,” thisapplication means removing at least 75%, at least 80%, at least 90%, atleast 95%, or about 100% of the contents of the ordinary volume of coloncontents. Antibiotic treatment may precede the colon-cleansing protocol.

If a patient has received an antibiotic for treatment of an infection,or if a patient has received an antibiotic as part of a specificpretreatment protocol, in one embodiment the antibiotic should bestopped in sufficient time to allow the antibiotic to be substantiallyreduced in concentration in the gut before the bacterial composition isadministered. In one embodiment, the antibiotic may be discontinued 1,2, or 3 days before the administration of the bacterial composition. Inone embodiment, the antibiotic may be discontinued 3, 4, 5, 6, or 7antibiotic half-lives before administration of the bacterialcomposition. In another embodiment, the antibiotic may be chosen so theconstituents in the bacterial composition have an MIC50 that is higherthan the concentration of the antibiotic in the gut.

MIC50 of a bacterial composition or the elements in the composition maybe determined by methods well known in the art. Reller et al.,Antimicrobial Susceptibility Testing: A Review of General Principles andContemporary Practices, Clinical Infectious Diseases 49(11):1749-1755(2009). In such an embodiment, the additional time between antibioticadministration and administration of the bacterial composition is notnecessary. If the pretreatment protocol is part of treatment of an acuteinfection, the antibiotic may be chosen so that the infection issensitive to the antibiotic, but the constituents in the bacterialcomposition are not sensitive to the antibiotic.

Routes of Administration

The bacterial compositions of the invention are suitable foradministration to mammals and non-mammalian animals in need thereof. Incertain embodiments, the mammalian subject is a human subject who hasone or more symptoms of a dysbiosis.

When the mammalian subject is suffering from a disease, disorder orcondition characterized by an aberrant microbiota, the bacterialcompositions described herein are suitable for treatment thereof. Insome embodiments, the mammalian subject has not received antibiotics inadvance of treatment with the bacterial compositions. For example, themammalian subject has not been administered at least two doses ofvancomycin, metronidazole and/or or similar antibiotic compound withinone week prior to administration of the therapeutic composition. Inother embodiments, the mammalian subject has not previously received anantibiotic compound in the one month prior to administration of thetherapeutic composition. In other embodiments, the mammalian subject hasreceived one or more treatments with one or more different antibioticcompounds and such treatment(s) resulted in no improvement or aworsening of symptoms.

In some embodiments, the gastrointestinal disease, disorder or conditionis diarrhea caused by C. difficile including recurrent C. difficileinfection, ulcerative colitis, colitis, Crohn's disease, or irritablebowel disease. Beneficially, the therapeutic composition is administeredonly once prior to improvement of the disease, disorder or condition. Insome embodiments the therapeutic composition is administered atintervals greater than two days, such as once every three, four, five orsix days, or every week or less frequently than every week. Or thepreparation may be administered intermittently according to a setschedule, e.g., once a day, once weekly, or once monthly, or when thesubject relapses from the primary illness. In another embodiment, thepreparation may be administered on a long-term basis to subjects who areat risk for infection with or who may be carriers of these pathogens,including subjects who will have an invasive medical procedure (such assurgery), who will be hospitalized, who live in a long-term care orrehabilitation facility, who are exposed to pathogens by virtue of theirprofession (livestock and animal processing workers), or who could becarriers of pathogens (including hospital workers such as physicians,nurses, and other health care professionals).

In embodiments, the bacterial composition is administered enterically.This preferentially includes oral administration, or by an oral or nasaltube (including nasogastric, nasojejunal, oral gastric, or oraljejunal). In other embodiments, administration includes rectaladministration (including enema, suppository, or colonoscopy). Thebacterial composition may be administered to at least one region of thegastrointestinal tract, including the mouth, esophagus, stomach, smallintestine, large intestine, and rectum. In some embodiments it isadministered to all regions of the gastrointestinal tract. The bacterialcompositions may be administered orally in the form of medicaments suchas powders, capsules, tablets, gels or liquids. The bacterialcompositions may also be administered in gel or liquid form by the oralroute or through a nasogastric tube, or by the rectal route in a gel orliquid form, by enema or instillation through a colonoscope or by asuppository.

If the composition is administered colonoscopically and, optionally, ifthe bacterial composition is administered by other rectal routes (suchas an enema or suppository) or even if the subject has an oraladministration, the subject may have a colon-cleansing preparation. Thecolon-cleansing preparation can facilitate proper use of the colonoscopeor other administration devices, but even when it does not serve amechanical purpose it can also maximize the proportion of the bacterialcomposition relative to the other organisms previously residing in thegastrointestinal tract of the subject. Any ordinarily acceptablecolon-cleansing preparation may be used such as those typically providedwhen a subject undergoes a colonoscopy.

Dosages and Schedule for Administration

In some embodiments the bacteria and bacterial compositions are providedin a dosage form. In some embodiments the dosage form is designed foradministration of at least one OTU or combination thereof disclosedherein, wherein the total amount of bacterial composition administeredis selected from 0.1 ng to 10 g, 10 ng to 1 g, 100 ng to 0.1 g, 0.1 mgto 500 mg, 1 mg to 100 mg, or from 10-15 mg. In some embodiments thebacterial composition is consumed at a rate of from 0.1 ng to 10 g aday, 10 ng to 1 g a day, 100 ng to 0.1 g a day, 0.1 mg to 500 mg a day,1 mg to 100 mg a day, or from 10-15 mg a day, or more.

In some embodiments the treatment period is at least 1 day, at least 2days, at least 3 days, at least 4 days, at least 5 days, at least 6days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 1 month, at least 2 months, at least 3 months, at least4 months, at least 5 months, at least 6 months, or at least 1 year. Insome embodiments the treatment period is from 1 day to 1 week, from 1week to 4 weeks, from 1 month, to 3 months, from 3 months to 6 months,from 6 months to 1 year, or for over a year.

In one embodiment, from 10⁵ and 10 microorganisms total may beadministered to the patient in a given dosage form. In one mode, aneffective amount may be provided in from 1 to 500 ml or from 1 to 500grams of the bacterial composition having from 10⁷ to 10¹¹ bacteria perml or per gram, or a capsule, tablet or suppository having from 1 mg to1000 mg lyophilized powder having from 10⁷ to 10¹¹ bacteria. Thosereceiving acute treatment may receive higher doses than those who arereceiving chronic administration (such as hospital workers or thoseadmitted into long-term care facilities).

Any of the preparations described herein may be administered once on asingle occasion or on multiple occasions, such as once a day for severaldays or more than once a day on the day of administration (includingtwice daily, three times daily, or up to five times daily). Or thepreparation may be administered intermittently according to a setschedule, e.g., once weekly, once monthly, or when the patient relapsesfrom the primary illness. In another embodiment, the preparation may beadministered on a long-term basis to individuals who are at risk forinfection with or who may be carriers of these pathogens, includingindividuals who will have an invasive medical procedure (such assurgery), who will be hospitalized, who live in a long-term care orrehabilitation facility, who are exposed to pathogens by virtue of theirprofession (livestock and animal processing workers), or who could becarriers of pathogens (including hospital workers such as physicians,nurses, and other health care professionals).

Patient Selection

Particular bacterial compositions may be selected for individualpatients or for patients with particular profiles. For example, 16Ssequencing may be performed for a given patient to identify the bacteriapresent in his or her microbiota. The sequencing may either profile thepatient's entire microbiome using 16S sequencing (to the family, genera,or species level), a portion of the patient's microbiome using 16Ssequencing, or it may be used to detect the presence or absence ofspecific candidate bacteria that are biomarkers for health or aparticular disease state, such as markers of multi-drug resistantorganisms or specific genera of concern such as Escherichia. Based onthe biomarker data, a particular composition may be selected foradministration to a patient to supplement or complement a patient'smicrobiota in order to restore health or treat or prevent disease. Inanother embodiment, patients may be screened to determine thecomposition of their microbiota to determine the likelihood ofsuccessful treatment.

Combination Therapy

The bacterial compositions may be administered with other agents in acombination therapy mode, including anti-microbial agents andprebiotics. Administration may be sequential, over a period of hours ordays, or simultaneous.

In one embodiment, the bacterial compositions are included incombination therapy with one or more anti-microbial agents, whichinclude anti-bacterial agents, anti-fungal agents, anti-viral agents andanti-parasitic agents.

Anti-bacterial agents include cephalosporin antibiotics (cephalexin,cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole,cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics(cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracyclineantibiotics (tetracycline, minocycline, oxytetracycline, anddoxycycline); penicillin antibiotics (amoxicillin, ampicillin,penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); and carbapenem antibiotics (ertapenem, doripenem,imipenem/cilastatin, and meropenem).

Anti-viral agents include Abacavir, Acyclovir, Adefovir, Amprenavir,Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol,Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine,Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine,Lamivudine, Lopinavir Maraviroc, MK-2048, Nelfinavir, Nevirapine,Penciclovir, Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine,Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine,Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir,Zalcitabine, Zanamivir and Zidovudine.

Examples of antifungal compounds include, but are not limited to polyeneantifungals such as natamycin, rimocidin, filipin, nystatin,amphotericin B, candicin, and hamycin; imidazole antifungals such asmiconazole, ketoconazole, clotrimazole, econazole, omoconazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, and tioconazole; triazole antifungals suchas fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole,voriconazole, terconazole, and albaconazole; thiazole antifungals suchas abafungin; allylamine antifungals such as terbinafine, naftifine, andbutenafine; and echinocandin antifungals such as anidulafungin,caspofungin, and micafungin. Other compounds that have antifungalproperties include, but are not limited to polygodial, benzoic acid,ciclopirox, tolnaftate, undecylenic acid, flucytosine or5-fluorocytosine, griseofulvin, and haloprogin.

In one embodiment, the bacterial compositions are included incombination therapy with one or more corticosteroids, mesalazine,mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressivedrugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone,methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines, and combinations thereof.

A prebiotic is a selectively fermented ingredient that allows specificchanges, both in the composition and/or activity in the gastrointestinalmicrobiota that confers benefits upon host well-being and health.Prebiotics may include complex carbohydrates, amino acids, peptides, orother essential nutritional components for the survival of the bacterialcomposition. Prebiotics include, but are not limited to, amino acids,biotin, fructooligosaccharide, galactooligosaccharides, inulin,lactulose, mannan oligosaccharides, oligofructose-enriched inulin,oligofructose, oligodextrose, tagatose, trans-galactooligosaccharide,and xylooligosaccharides.

Methods for Characterization of Bacterial Compositions

In certain embodiments, provided are methods for testing certaincharacteristics of bacterial compositions. For example, the sensitivityof bacterial compositions to certain environmental variables isdetermined, e.g., in order to select for particular desirablecharacteristics in a given composition, formulation and/or use. Forexample, the constituents in the bacterial composition may be tested forpH resistance, bile acid resistance, and/or antibiotic sensitivity,either individually on a constituent-by-constituent basis orcollectively as a bacterial composition comprised of multiple bacterialconstituents (collectively referred to in this section as bacterialcomposition).

pH Sensitivity Testing. If a bacterial composition will be administeredother than to the colon or rectum (i.e., through, for example, but notlimited to, an oral route), optionally testing for pH resistanceenhances the selection of bacterial compositions that will survive atthe highest yield possible through the varying pH environments of thedistinct regions of the GI tract. Understanding how the bacterialcompositions react to the pH of the GI tract also assists informulation, so that the number of bacteria in a dosage form can beincreased if beneficial and/or so that the composition may beadministered in an enteric-coated capsule or tablet or with a bufferingor protective composition. As the pH of the stomach can drop to a pH of1 to 2 after a high-protein meal for a short time before physiologicalmechanisms adjust it to a pH of 3 to 4 and often resides at a resting pHof 4 to 5, and as the pH of the small intestine can range from a pH of 6to 7.4, bacterial compositions can be prepared that survive thesevarying pH ranges (specifically wherein at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% of thebacteria can survive gut transit times through various pH ranges). Thismay be tested by exposing the bacterial composition to varying pH rangesfor the expected gut transit times through those pH ranges. Therefore,as a nonlimiting example only, 18-hour cultures of bacterialcompositions may be grown in standard media, such as gut microbiotamedium (“GMM”, see Goodman et al., Extensive personal human gutmicrobiota culture collections characterized and manipulated ingnotobiotic mice, PNAS 108(15):6252-6257 (2011)) or anotheranimal-products-free medium, with the addition of pH adjusting agentsfor a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1 hour, a pH of 4to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5 to 3 hours. Analternative method for testing stability to acid is described in U.S.Pat. No. 4,839,281. Survival of bacteria may be determined by culturingthe bacteria and counting colonies on appropriate selective ornon-selective media.

Bile Acid Sensitivity Testing. Additionally, in some embodiments,testing for bile-acid resistance enhances the selection of bacterialcompositions that will survive exposures to bile acid during transitthrough the GI tract. Bile acids are secreted into the small intestineand can, like pH, affect the survival of bacterial compositions. Thismay be tested by exposing the bacterial compositions to bile acids forthe expected gut exposure time to bile acids. For example, bile acidsolutions may be prepared at desired concentrations using 0.05 mM Trisat pH 9 as the solvent. After the bile acid is dissolved, the pH of thesolution may be adjusted to 7.2 with 10% HCl. Bacterial compositions maybe cultured in 2.2 ml of a bile acid composition mimicking theconcentration and type of bile acids in the patient, 1.0 ml of 10%sterile-filtered feces media and 0.1 ml of an 18-hour culture of thegiven strain of bacteria. Incubations may be conducted for from 2.5 to 3hours or longer. An alternative method for testing stability to bileacid is described in U.S. Pat. No. 4,839,281. Survival of bacteria maybe determined by culturing the bacteria and counting colonies onappropriate selective or non-selective media.

Antibiotic Sensitivity Testing. As a further optional sensitivity test,bacterial compositions may be tested for sensitivity to antibiotics. Inone embodiment, bacterial compositions may be chosen so that thebacterial constituents are sensitive to antibiotics such that ifnecessary they can be eliminated or substantially reduced from thepatient's gastrointestinal tract by at least one antibiotic targetingthe bacterial composition.

Adherence to Gastrointestinal Cells. The bacterial compositions mayoptionally be tested for the ability to adhere to gastrointestinalcells. A method for testing adherence to gastrointestinal cells isdescribed in U.S. Pat. No. 4,839,281.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments and should not be construed to limit the scope. The skilledartisan readily recognizes that many other embodiments are encompassed.All publications and patents cited in this disclosure are incorporatedby reference in their entirety. To the extent the material incorporatedby reference contradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are priorart.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to beobtained. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.

Examples

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for. Examples of the techniquesand protocols described herein with regard to therapeutic compositionscan be found in, e.g., Remington's Pharmaceutical Sciences, 16thedition, Osol, A. (ed), 1980.

Example 1. Construction of Binary Pairs in a High-Throughput 96-WellFormat

To allow high-throughput screening of binary pairs, vials of −80° C.glycerol stock banks were thawed and diluted to 1e8 CFU/mL. Each strainwas then diluted 10× (to a final concentration of 1e7 CFU/mL of eachstrain) into 200 uL of PBS+15% glycerol in the wells of a 96-well plate.Plates were then frozen at −80° C. When needed, plates were removed from−80° C. and thawed at room temperature under anaerobic conditions whentesting in a CivSim with Clostridium difficile.

Example 2. Construction of Ternary Combinations in a High-Throughput96-Well Format

To allow high-throughput screening of ternary combinations, vials of−80° C. glycerol stock banks were thawed and diluted to 1e8 CFU/mL. Eachstrain was then diluted 10× (to a final concentration of 1e7 CFU/mL ofeach strain) into 200 uL of PBS+15% glycerol in the wells of a 96-wellplate. Plates were then frozen at −80° C. When needed for the assay,plates were removed from −80° C. and thawed at room temperature underanaerobic conditions when testing in a CivSim with Clostridiumdifficile.

Example 3. Construction of a CivSim Assay to Screen for Ecobiotic™Compositions Inhibitory to the Growth of Clostridium difficile

An overnight culture of Clostridium difficile was grown under anaerobicconditions in SweetB-FosIn or other suitable media for the growth of C.difficile. SweetB-FosIn is a complex media composed of brain heartinfusion, yeast extract, cysteine, cellobiose, maltose, soluble starch,and fructooligosaccharides/inulin, and hemin, and is buffered with MOPs.After 24 hr of growth the culture was diluted 100,000 fold into acomplex media such as SweetB-FosIn which is suitable for the growth of awide variety of anaerobic bacterial species. The diluted C. difficilemixture was then aliquoted to wells of a 96-well plate (180 uL to eachwell). 20 uL of a unique binary pair of potential inhibitory species wasthen added to each well at a final concentration of 1e6 CFU/mL of eachspecies. Alternatively the assay can be tested with binary pairs atdifferent initial concentrations (1e9 CFU/mL, 1e8 CFU/mL, 1e7 CFU/mL,1e5 CFU/mL, 1e4 CFU/mL, 1e3 CFU/mL, 1e2 CFU/mL). Control wells onlyinoculated with C. difficile were included for a comparison to thegrowth of C. difficile without inhibition. Additional wells were usedfor controls that either inhibit or do not inhibit the growth of C.difficile. One example of a positive control that inhibits growth was acombination of Blautia producta, Clostridium bifermentans andEscherichia coli. One example of a control that shows reduced inhibitionof C. difficile growth as a combination of Bacteroides thetaiotaomicron,Bacteroides ovatus and Bacteroides vulgatus. Plates were wrapped withparafilm and incubated for 24 hr at 37° C. under anaerobic conditions.After 24 hr the wells containing C. difficile alone were seriallydiluted and plated to determine titer. The 96-well plate was then frozenat −80 C before quantifying C. difficile by qPCR assay.

Example 4. Construction of a CivSim Assay to Screen for BacterialCompositions that Produce Diffusible Products Inhibitory to the Growthof Clostridium difficile Using a Filter Insert

The CivSim assay described above was modified by using a 0.22 uM filterinsert (Millipore™ MultiScreen™ 96-Well Assay Plates—Item MAGVS2210) in96-well format to physically separate C. difficile from the bacterialcompositions. The C. difficile was aliquoted into the 96-well platewhile the bacterial compositions were aliquoted into media on the filteroverlay. The nutrient media as in contact on both sides of the 0.22 uMfilter, allowing exchange of nutrients, small molecules and manymacromolecules (e.g., bacteriocins, cell-surface proteins, orpolysaccharides) by diffusion. In this embodiment, after 24 hrincubation, the filter insert containing the bacterial compositions wasremoved. The plate containing C. difficile was then transferred to a96-well plate reader suitable for measuring optical density (OD) at 600nm. The growth of C. difficile in the presence of different bacterialcompositions was compared based on the OD measurement.

Example 5. Construction of a CivSim Assay to Screen for BacterialCompositions Inhibitory to the Growth of Clostridium difficile UsingClostridium difficile Selective Media for Quantification

The CivSim assay described above can be modified to determine final C.difficile titer by serially diluting and plating to C. difficileselective media (Bloedt et al 2009) such as CCFA (cycloserine cefoxitinfructose agar, Anaerobe Systems), CDSA (Clostridium difficile selectiveagar, which is cycloserine cefoxitin mannitol agar, Becton Dickinson).

Example 6. Quantification of C. difficile Using Quantitative PCR (qPCR)Standard Curve Preparation

The standard curve was generated from a well on each assay platecontaining only pathogenic C. difficile grown in SweetB+FosIn media asprovided herein and quantified by selective spot plating. Serialdilutions of the culture were performed in sterile phosphate-bufferedsaline. Genomic DNA was extracted from the standard curve samples alongwith the other wells.

Genomic DNA Extraction

Genomic DNA was extracted from 5 μl of each sample using a dilution,freeze/thaw, and heat lysis protocol. 5 μL of thawed samples were addedto 45 μL of UltraPure water (Life Technologies, Carlsbad, Calif.) andmixed by pipetting. The plates with diluted samples were frozen at −20°C. until use for qPCR which includes a heated lysis step prior toamplification. Alternatively the genomic DNA could be isolated using theMo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), Mo Bio Powersoil® DNA Isolation Kit (MoBio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

qPCR Composition and Conditions

The qPCR reaction mixture contained 1× SsoAdvanced Universal ProbesSupermix, 900 nM of Wr-tcdB-F primer (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG(SEQ ID NO: 1916), IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R primer(CATGCTTTTTTAGTTTCTGGATTGAA (SEQ ID NO: 1917), IDT, Coralville, Iowa),250 nM of Wr-tcdB-P probe (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQID NO: 1918), Life Technologies, Grand Island, N.Y.), and MolecularBiology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 μl(Primers adapted from: Wroblewski, D. et al., Rapid MolecularCharacterization of Clostridium difficile and Assessment of Populationsof C. difficile in Stool Specimens, Journal of Clinical Microbiology47:2142-2148 (2009)). This reaction mixture was aliquoted to wells of aHard-shell Low-Profile Thin Wall 96-well Skirted PCR Plate (BioRad,Hercules, Calif.). To this reaction mixture, 2 μl of diluted, frozen,and thawed samples were added and the plate sealed with a Microseal ‘B’Adhesive Seal (BioRad, Hercules, Calif.). The qPCR was performed on aBioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System(BioRad, Hercules, Calif.). The thermocycling conditions were 95° C. for15 minutes followed by 45 cycles of 95° C. for 5 seconds, 60° C. for 30seconds, and fluorescent readings of the FAM channel. Alternatively, theqPCR could be performed with other standard methods known to thoseskilled in the art.

Data Analysis

The Cq value for each well on the FAM channel was determined by the CFXManager™ 3.0 software. The log₁₀ (cfu/mL) of C. difficile eachexperimental sample was calculated by inputting a given sample's Cqvalue into a linear regression model generated from the standard curvecomparing the Cq values of the standard curve wells to the known log₁₀(cfu/mL) of those samples. The log inhibition was calculated for eachsample by subtracting the log₁₀ (cfu/mL) of C. difficile in the samplefrom the log₁₀ (cfu/mL) of C. difficile in the sample on each assayplate used for the generation of the standard curve that has noadditional bacteria added. The mean log inhibition was calculated forall replicates for each composition.

A histogram of the range and standard deviation of each composition wasplotted. Ranges or standard deviations of the log inhibitions that weredistinct from the overall distribution were examined as possibleoutliers. If the removal of a single log inhibition datum from one ofthe binary pairs that were identified in the histograms would bring therange or standard deviation in line with those from the majority of thesamples, that datum was removed as an outlier, and the mean loginhibition was recalculated.

The pooled variance of all samples evaluated in the assay was estimatedas the average of the sample variances weighted by the sample's degreesof freedom. The pooled standard error was then calculated as the squareroot of the pooled variance divided by the square root of the number ofsamples. Confidence intervals for the null hypothesis were determined bymultiplying the pooled standard error to the z score corresponding to agiven percentage threshold. Mean log inhibitions outside the confidenceinterval were considered to be inhibitory if positive or stimulatory ifnegative with the percent confidence corresponding to the interval used.Samples with mean log inhibition greater than the 99% confidenceinterval (C.I) of the null hypothesis are reported as ++++, those with a95%<C.I.<99% as +++, those with a 90%<C.I.<95% as ++, those with a80%<C.I.<90% as + while samples with mean log inhibition less than the99% confidence interval (C.I) of the null hypothesis are reported as−−−−,those with a 95%<C.I.<99% as −−−, those with a 90%<C.I.<95% as −−,those with a 80%<C.I.<90% as −.

Many binary pairs inhibit C. difficile Table 3. 622 of 989 combinationsshow inhibition with a confidence interval>80%; 545 of 989 with aC.I.>90%; 507 of 989 with a C.I.>95%; 430 of 989 with a C.I. of >99%.Non-limiting but exemplary binary pairs include those with mean logreduction greater than 0.366, e.g. Allistipes shahii paired with Blautiaproducta, Clostridium hathaweyi, or Colinsella aerofaciens, orClostidium mayombei paired with C. innocuum, C. tertium, Colinsellaaerofaciens, or any of the other 424 combinations shown in Table 5.Equally important, the CivSim assay describes binary pairs that do noteffectively inhibit C. difficile. 188 of 989 combinations promote growthwith >80% confidence; 52 of 989 show a lack of inhibition with >90%confidence; 22 of 989 show a lack of inhibition with >95% confidence; 3of 989, including B. producta combined with Coprococcus catus, Alistipesshahii combined with Dorea formicigenerans, and Eubacterium rectalecombined with Roseburia intestinalis, show a lack of inhibitionwith >99% confidence. 249 of 989 combinations are neutral in the assay,meaning they neither promote nor inhibit C. difficile growth to thelimit of measurement.

Ternary combinations with mean log inhibition greater than 0.312 arereported as ++++ (≥99% confidence interval (C.I.) of the nullhypothesis), those with mean log inhibition between 0.221 and 0.312 as+++ (95%<C.I.<99%), those with mean log inhibition between 0.171 and0.221 as ++ (90%<C.I.<95%), those with mean log inhibition between 0.113and 0.171 as + (80%<C.I.<90%), those with mean log inhibition between−0.113 and −0.171 as −(80%<C.I.<90%), those with mean log inhibitionbetween −0.171 and −0.221 as −− (90%<C.I.<95%), those with mean loginhibition between −0.221 and −0.312 as −−− (95%<C.I.<99%), and thosewith mean log inhibition less than −0.312 as −−−− (99%<C.I.).

The CivSim shows that many ternary combinations inhibit C. difficile. 39of 56 combinations show inhibition with a confidence interval>80%; 36 of56 with a C.I.>90%; 36 of 56 with a C.I.>95%; 29 of 56 with a C.I.of >99%. Non-limiting but exemplary ternary combinations include thosewith mean log reduction greater than 0.171, e.g. any combination shownin Table 4 with a score of ++++, such as Colinsella aerofaciens,Coprococcus comes, and Blautia producta. Equally important, the CivSimassay describes ternary combinations that do not effectively inhibit C.difficile. 5 of 56 combinations promote growth with >80% confidence; 2of 56 promote growth with >90% confidence; 1 of 56, Coprococcus comes,Clostridium symbiosum and Eubacterium rectale, promote growth with >95%confidence. 12 of 56 combinations are neutral in the assay, meaning theyneither promote nor inhibit C. difficile growth to the limit ofmeasurement.

Example P1. Full 16S Sequencing to Determine Operational Taxonomic Unit(OTU) Genomic DNA Extraction

Genomic DNA is extracted from pure microbial cultures using a hotalkaline lysis method. 2 μl of microbial culture is added to 18 μl ofLysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the mixture is incubated at95° C. for 30 minutes. Subsequently, the samples are cooled to 4° C. andneutralized by the addition of 20 μl of Neutralization Buffer (40 mMTris-HCl) and then diluted 10-fold in Elution Buffer (10 mM Tris-HCl).Alternatively, genomic DNA is extracted from pure microbial culturesusing commercially available kits such as the Mo Bio Ultraclean®Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) orby standard methods known to those skilled in the art.

PCR

To amplify bacterial 16S rDNA, 2 μl of extracted gDNA is added to a 20μl final volume PCR reaction. The PCR reaction also contains 1×HotMasterMix (5PRIME, Gaithersburg, Md.), 250 nM of 27f primer(AGRGTTTGATCMTGGCTCAG (SEQ ID NO: 1919), IDT, Coralville, Iowa), and 250nM of 1492r primer (TACGGYTACCTTGTTAYGACTT (SEQ ID NO: 1920), IDT,Coralville, Iowa), with Molecular Biology Grade Water (Mo BioLaboratories, Carlsbad, Calif.) for the balance of the volume.Alternatively, other universal bacterial primers or thermostablepolymerases known to those skilled in the art are used.

The PCR performed on commercially available thermocyclers such as aBioRad MyCycler™ Thermal Cycler (BioRad, Hercules, Calif.). Thereactions are run at 94° C. for 2 minutes followed by 30 cycles of 94°C. for 30 seconds, 51° C. for 30 seconds, and 68° C. for 1 minute 30seconds, followed by a 7 minute extension at 72° C. and an indefinitehold at 4° C. Following PCR, gel electrophoresis of a portion of thereaction products is used to confirm successful amplification of a ˜1.5kb product.

PCR Cleanup

To remove nucleotides and oligonucleotides from the PCR products, 1 μlof HT ExoSap-IT (Affymetrix, Santa Clara, Calif.) is added to 2.5 μl ofPCR product followed by a 15 minute incubation at 37° C. and then a 15minute inactivation at 80° C.

Sanger Sequencing

For each sample, two sequencing reactions are performed, one using eachprimer: 27f and 1492r. 40 ng of ExoSap-IT-cleaned PCR products are mixedwith 25 pmol of sequencing primer and Molecular Biology Grade Water (MoBio Laboratories, Carlsbad, Calif.) to 15 μl total volume. This reactionis submitted to a commercial sequencing organization such as Genewiz(South Plainfield, N.J.) for Sanger sequencing.

Example P2. V4 16S Sequencing to Determine Operational Taxonomic Unit(OTU) Genomic DNA Extraction

Genomic DNA is extracted from pure microbial cultures using a hotalkaline lysis method. 2 μl of microbial culture is added to 18 μl ofLysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the mixture is incubated at95° C. for 30 minutes. Subsequently, the samples are cooled to 4° C. andneutralized by the addition of 18 μl of Neutralization Buffer (40 mMTris-HCl) and then diluted 10-fold in Elution Buffer (10 mM Tris-HCl).Alternatively, genomic DNA is extracted from pure microbial culturesusing commercially available kits such as the Mo Bio Ultraclean®Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) orby standard methods known to those in skilled in the art.

PCR

To amplify the V4 region of bacterial 16S rDNA, 2 μl of extracted gDNAis added to a 20 μl final volume PCR reaction. The PCR reaction alsocontains 1× HotMasterMix (5PRIME, Gaithersburg, Md.), 200 nM ofV4_515f_adapt (AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 1921), IDT, Coralville, Iowa), and 200 nM of barcoded806rbc (CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO:1922)_12bpGolayBarcode_AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO:1923), IDT, Coralville, Iowa), with Molecular Biology Grade Water (MoBio Laboratories, Carlsbad, Calif.) for the balance of the volume. Theseprimers incorporate adapters for Illumina sequencing by synthesis.Optionally, identical replicate, triplicate, or quadruplicate reactionsmay be performed. Alternatively other universal bacterial primers orthermostable polymerases known to those skilled in the art are used.

The PCR performed on commercially available thermocyclers such as aBioRad MyCycler™ Thermal Cycler (BioRad, Hercules, Calif.). Thereactions are run at 94° C. for 3 minutes followed by 25 cycles of 94°C. for 45 seconds, 50° C. for 1 minute, and 72° C. for 1 minute 30seconds, followed by a 10 minute extension at 72° C. and a indefinitehold at 4° C. Following PCR, gel electrophoresis of a portion of thereaction products is used to confirm successful amplification of a ˜0.4kb product.

PCR Cleanup

To remove nucleotides and oligonucleotides from the PCR products, theentire remaining volume of the PCR, or of the multiple PCRs, is cleanedup using the Mo Bio Ultraclean®-htp 96 Well PCR Clean-up Kit (Mo BioLaboratories, Carlsbad, Calif.) according to the manufacturer'sinstructions or other commercially available kits such as the QIAquick96 PCR Purification Kit (QIAGEN, Valencia, Calif.).

DNA Quantification & Pooling

The cleaned PCR products are quantified using the Quant-iT™ PicoGreen®dsDNA Assay Kit (Life Technologies, Grand Island, N.Y.) according to themanufacturer's instructions. Following quantification, the barcodedcleaned PCR products are combined such that each distinct PCR product isat an equimolar ratio to create a prepared Illumina library.

Illumina Sequencing

The prepared library is sequenced on Illumina HiSeq or MiSeq sequencers(Illumina, San Diego, Calif.) with cluster generation, templatehybridization, iso-thermal amplification, linearization, blocking anddenaturization and hybridization of the sequencing primers performedaccording to the manufacturer's instructions. 16SV4SeqFw(TATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 1924)), 16SV4SeqRev(AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 1923)), and 16SV4Index(ATTAGAWACCCBDGTAGTCCGGCTGACTGACT (SEQ ID NO: 1925)) (IDT, Coralville,Iowa) are used for sequencing. This sequencing can optionally beperformed by a contract research organization such as Metanome (Houston,Tex.), Ambry Genetics (Aliso Viejo, Calif.), Edge Bio (Gaithersburg,Md.), or Covance (Princeton, N.J.).

Taxonomic Assignment to Sequence Read Data

Nucleic acid sequences are analyzed and taxonomic and phylogeneticassignments of specific OTUs are made using sequence similarity andphylogenetic methods that are well known to those skilled in the art,including but not limited to maximum likelihood phylogeneticreconstruction (see e.g. Liu K, Linder C R, and Warnow T. 2011. RAxMLand FastTree: Comparing Two Methods for Large-Scale Maximum LikelihoodPhylogeny Estimation. PLoS ONE 6: e27731. McGuire G, Denham M C, andBalding D J. 2001. Models of sequence evolution for DNA sequencescontaining gaps. Mol. Biol. Evol 18: 481-490. Wróbel B. 2008.Statistical measures of uncertainty for branches in phylogenetic treesinferred from molecular sequences by using model-based methods. J. Appl.Genet. 49: 49-67.) From these taxonomic assignments OTUs in the datasetare defined. The certainty of the OTU call is defined based on the OTU'ssequence similarity to a reference nucleic acid sequence and theproximity of the OTU sequence relative to one or more referencesequences in the phylogeny. The specificity of an OTU's taxonomic andphlylogenetic assignment determines whether the match is assigned at thelevel of Family, Genus, Species, or Strain, and the confidence of thisassignment is determined based on the position of bootstrap supportedbranches in the reference phylogenetic tree relative to the placement ofthe OTU sequence being interrogated.

Example P3. Construction of an In Vitro Assay to Screen for Combinationsof Microbes Inhibitory to the Growth of Pathogenic E. coli

The in vitro assay is used to screen for combinations of bacteriainhibitory to the growth of E. coli by modifying the media used forgrowth of the pathogen inoculum. One of several choices of media is usedfor growth of the pathogen such as Reinforced Clostridial Media (RCM),Brain Heart Infusion Broth (BHI) or Luria Bertani Broth (LB) (also knownas Lysogeny Broth). E. coli is quantified by using alternative selectivemedia specific for E. coli or using qPCR probes specific for thepathogen. For example, aerobic growth on MacConkey lactose mediumselects for enteric Gram negatives, including E. coli. qPCR is conductedusing probes specific for the shiga toxin of pathogenic E. coli.

Example P4. Construction of an In Vitro Assay to Screen for Combinationsof Microbes Inhibitory to the Growth of Vancomycin-ResistantEnterococcus (VRE)

The in vitro assay is used to screen for combinations of bacteriainhibitory to the growth of Vancomycin-Resistant Enterococcus spp. (VRE)by modifying the media used for growth of the pathogen inoculum. Severalchoices of media are used for growth of the pathogen such as ReinforcedClostridial Media (RCM), Brain Heart Infusion Broth (BHI) or LuriaBertani Broth (LB). VRE is quantified by using alternative selectivemedia specific for VRE or using qPCR probes specific for the pathogen.For example, m-Enterococcus agar containing sodium azide is selectivefor Enterococcus spp. and a small number of other species. Probesspecific to the van genes conferring vancomycin resistance are used inthe qPCR.

Example P5. Testing of Bacterial Composition Against Salmonella

The in vitro assay is used to screen for combinations of bacteriainhibitory to the growth of Salmonella spp. by modifying the media usedfor growth of the pathogen inoculum. Several choices of media are usedfor growth of the pathogen such as Reinforced Clostridial Media (RCM),Brain Heart Infusion Broth (BHI) or Luria Bertani Broth (LB). Salmonellaspp. are quantified by using alternative selective media specific forSalmonella spp. or using qPCR probes specific for the pathogen. Forexample, MacConkey agar is used to select for Salmonella spp. and theinvA gene is targeted with qPCR probes; this gene encodes an invasionprotein carried by many pathogenic Salmonella spp. and is used ininvading eukaryotic cells.

Example P6. Method of Preparing the Bacterial Composition forAdministration to a Subject

Two strains for the bacterial composition are independently cultured andmixed together before administration. Both strains are independently begrown at 37° C., pH 7, in a GMM or other animal-products-free medium,pre-reduced with 1 g/L cysteine□HCl. After each strain reaches asufficient biomass, it is preserved for banking by adding 15% glyceroland then frozen at −80° C. in 1 ml cryotubes.

Each strain is then be cultivated to a concentration of 10¹⁰ CFU/mL,then concentrated 20-fold by tangential flow microfiltration; the spentmedium is exchanged by diafiltering with a preservative mediumconsisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphatebuffer, or other suitable preservative medium. The suspension isfreeze-dried to a powder and titrated.

After drying, the powder is blended with microcrystalline cellulose andmagnesium stearate and formulated into a 250 mg gelatin capsulecontaining 10 mg of lyophilized powder (10⁸ to 10¹¹ bacteria), 160 mgmicrocrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesiumstearate.

Example P7. Method of Treating a Subject with a Bacterial Composition

A patient has suffered from recurrent bouts of C. difficile. In the mostrecent acute phase of illness, the patient is treated with an antibioticsufficient to ameliorate the symptoms of the illness. In order toprevent another relapse of C. difficile, the patient is administered oneof the present bacterial compositions. Specifically, the patient isadministered Bacillus circulans and Roseburia inulinivorans at a dose of10⁸ bacteria total in a lyophilized form, specifically in a 250 mggelatin capsule containing 10 mg of lyophilized bacteria, 160 mgmicrocrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesiumstearate. The patient takes the capsule by mouth and resumes a normaldiet after 4, 8, 12, or 24 hours. In another embodiment, the patient maytake the capsule by mouth before, during, or immediately after a meal.

Feces is collected before and at 1 day, 3 days, 1 week, and 1 monthafter administration. The presence of C. difficile is found in the fecesbefore administration of the bacterial composition, but fecescollections after administration show reducing (such as at least 50%less, 60%, 70%, 80%, 90%, or 95%) to no detectable levels of C.difficile, as measured by qPCR, as described above. ELISA for toxinprotein or traditional microbiological identification techniques mayalso be used.

As another measure of patient success, a positive response may bedefined as absence of diarrhea, which itself is defined as 3 or moreloose or watery stools per day for at least 2 consecutive days or 8 ormore loose or watery stools in 48 hours, or persisting diarrhea (due toother causes) with repeating (three times) negative stool tests fortoxins of C. difficile.

Treatment failure is defined as persisting diarrhea with a positive C.difficile toxin stool test or no reduction in levels of C. difficile, asmeasured by qPCR sequencing. ELISA or traditional microbiologicalidentification techniques may also be used.

Example P8. Method of Treating a Subject with a Bacterial Composition

A patient has suffered from recurrent bouts of C. difficile. In the mostrecent acute phase of illness, the patient is treated with an antibioticsufficient to ameliorate the symptoms of the illness. In order toprevent another relapse of C. difficile, the patient is administered oneof the present bacterial compositions. Specifically, the patient isadministered a bacterial composition containing two bacterial types fromTable 1 or SEQ ID NOs. 1-1,864, or a combination from Table 2, at a doseof 10⁸ bacteria total in a lyophilized form formulated in an entericcoated capsule. Example of the patient or samples derived from thepatient is expected to demonstrate at least one measure of success asdescribed herein (reducing levels of C. difficile as measured by qPCR,ELISA, or traditional microbiological identification; absence ofdiarrhea; persisting diarrhea with repeating (three times) negativestool tests for toxins of C. difficile.

Other embodiments will be apparent to those skilled in the art fromconsideration of the specification and practice of the embodiments.Consider the specification and examples as exemplary only, with a truescope and spirit being indicated by the following claims.

Lengthy table referenced here US20210169949A1-20210610-T00001 Pleaserefer to the end of the specification for access instructions.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210169949A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A composition comprising a first species ofisolated bacterium and a second species of isolated bacterium, whereinthe first species and the second species are not identical, and whereinthe first species and the second species independently comprise a 16SrDNA sequence that is at least 97% identical to a 16S rDNA sequencepresent in a reference bacterium selected from Table 3 with the provisothat (i) if the first species is Clostridium innocuum, then the secondis not Clostridium orbiscindens, or (ii) if the first species isClostridium orbiscindens, then the second species is not Clostridiuminnocuum, and wherein the first species and the second species arecapable of inhibiting Clostridium difficile (C. difficile) growth asmeasured by a CivSim assay.
 2. The composition of claim 1, which furthercomprises an enteric coating.
 3. The composition of claim 1, wherein thefirst species and the second species are capable of inhibiting C.difficile growth in the CivSim assay with a confidence interval of >99%(++++).
 4. The composition of claim 1, wherein the first species and thesecond species comprise a combination of bacteria selected from Table 5.5. The composition of claim 1, wherein the first species, the secondspecies, or both are capable of forming a spore.
 6. The composition ofclaim 1, wherein the first species, the second species, or both are inthe form of spores.
 7. The composition of claim 1, which is formulatedfor populating the gastrointestinal tract of the subject.
 8. Thecomposition of claim 1, wherein the first species, the second species,or both are lyophilized.
 9. A method of treating or preventingrecurrence of Clostridium difficile infection in a subject in needthereof, comprising administering the composition of claim 1 to thesubject.
 10. A method of treating a dysbiosis in a subject in needthereof, comprising administering the composition of claim 1 to thesubject.
 11. A single dose unit comprising a first species of isolatedbacterium and a second species of isolated bacterium, wherein the firstspecies and the second species are capable of inhibiting Clostridiumdifficile (C. difficile) growth as measured by a CivSim assay and/orpopulating the gastrointestinal tract of a subject, and wherein (i) ifthe first species is Clostridium innocuum, then the second is notClostridium orbiscindens, or (ii) if the first species is Clostridiumorbiscindens, then the second species is not Clostridium innocuum.
 12. Akit comprising in one or more containers the composition of claim
 1. 13.A pharmaceutical formulation comprising the composition of claim
 1. 14.The pharmaceutical formulation of claim 13, further comprising anadditional agent.
 15. The pharmaceutical formulation of claim 14,wherein the additional agent is selected from an anti-bacterial agent,anti-fungal agent, anti-viral agent, anti-parasitic agent, orcombinations thereof.
 16. A method of producing a bacterial composition,comprising combining a first species of isolated bacterium and a secondspecies of isolated bacterium, wherein the first species and the secondspecies are not identical, wherein the first species and the secondspecies are capable of inhibiting Clostridium difficile (C. difficile)growth and/or populating the gastrointestinal tract of a subject, andwherein (i) if the first species is Clostridium innocuum, then thesecond is not Clostridium orbiscindens, or (ii) if the first species isClostridium orbiscindens, then the second species is not Clostridiuminnocuum.
 17. The method of claim 16, wherein the first species and thesecond species are lyophilized.